TDP-43 specific binding molecules

ABSTRACT

Provided are TAR DNA-binding protein of 43 kDa (TDP-43)-specific binding molecules including polypeptides such as human antibodies, as well as fragments, derivatives and variants thereof. Also provided are methods related to these TDP-43 specific binding molecules. Assays, kits, and solid supports related to TDP-43-specific binding molecules, including polypeptides such as, human antibodies are also disclosed. The TDP-43-specific binding molecule, antibody, immunoglobulin chain(s), as well as binding fragments, derivatives and variants thereof can be used in pharmaceutical and diagnostic compositions for TDP-43 targeted immunotherapy and diagnosis, respectively.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a divisional of U.S. application Ser. No.14/354,404, filed on Apr. 25, 2014, which is the National Stage ofInternational Application No. PCT/IB2012/002905, filed on Oct. 26, 2012,which claims the benefit of priority U.S. Application No. 61/553,113,filed on Oct. 28, 2011. The entire contents of each of these priorapplications is incorporated hereby by reference in their entirety.

FIELD OF THE INVENTION

The invention generally relates to novel TDP-43 (TAR DNA-binding proteinof 43 kDa)-specific binding molecules, such as antibodies, includingfragments, derivatives and variants thereof, that specifically recognizeTDP-43. In addition, the invention relates to pharmaceutical anddiagnostic compositions comprising such antibodies and otherTDP-43-specific binding molecules and their use to detect and identifyTDP-43 in plasma, cerebrospinal fluid, brain, and other samples, as wellas in therapeutic applications including for example, passivevaccination strategies for treating disorders related to aggregates orother aberrations in TDP-43 expression, such as, frontotemporal lobardegeneration and/or, amyotrophic lateral sclerosis, Alzheimer's disease,Parkinson's disease and other TDP-43 proteinopathies.

BACKGROUND OF THE INVENTION

Frontotemporal lobar degeneration (FTLD) is the second most common causeof dementia affecting individuals younger than 65 years; see, e.g.,McKhann et al., Arch. Neurol. 58 (2001), 1803; Forman et al., Ann.Neurol. 59 (2006), 952-62. On a cellular pathologic level, thecharacteristic lesions in the majority of FTLD brains are abnormalubiquitinated protein inclusions. The biochemical composition of theubiquitinated inclusions in the most common pathological form of FTLD,namely FTLD-U, remained unknown until 2006, when the TAR-DNA bindingprotein 43 (TDP-43) was identified as the major disease protein in themajority of sporadic and familial FTLD-U cases. Subsequently, theubiquitinated compact inclusions, characteristic for amyotrophic lateralsclerosis (ALS) were also found to be composed of TDP-43, therebyproviding evidence that both conditions are mechanistically linked andpart of spectrum of diseases which can be classified as TDP-43proteinopathies, see, e.g., Neumann et al., Science 314 (2006), 130-133.

Other than FTLD and ALS, TDP-43 is also known to accumulate in the nervecells and glial cells of ALS-Parkinsonism dementia complex of Guam,corticobasal degeneration, Dementia with Lewy bodies, Huntington'sdisease, Lewy body disease, motor neuron disease, frontotemporaldementia, frontotemporal lobar degeneration with ubiquitin-positiveinclusions, hippocampal sclerosis, inclusion body myopathy, inclusionbody myositis, Parkinson's disease, Parkinson's disease dementia,Parkinson-dementia complex in Kii peninsula and Pick's disease and thelike; see e.g., Lagier-Tourenne et al., Hum. Mol. Gen. 19 (2010),R46-64, which is herein incorporated by reference in its entirety. Thesediseases are collectively referred to as TDP-43 proteinopathies.Abnormal accumulation of TDP-43 is observed at the site of lesions ofeach disease which appears to imply involvement in the cause of nervedegeneration in these diseases. Increased cytoplasmic localization ofTDP-43 in brains and spinal cords of patients termed as “pre-inclusions”has been proposed to be an early event in TDP-43 proteinopathies, withthe implication of a possible pathogenic role in these diseases; see,e.g. Giordana et al., Brain Pathol. 20 (2010), 351-60. Consistently,increased cytoplasmic TDP-43 localization at presymptomatic stages hasbeen reported to be found in mice overexpressing wild type TDP-43; seee.g., Wils et al., Proc. Natl Acad. Sci. USA 107 (2010), 3858-63 as wellas in an acute rat model with adenovirus-mediated wild-type TDP-43expression; see, e.g., Tatom et al., Mol. Ther. 17 (2009), 607-613.Commercially available monoclonal murine antibodies are primarily usedin the studies on TDP-43 and to conduct pathological diagnosis of TDP-43proteinopathies. A monoclonal murine anti-TDP-43 antibody, whichrecognizes the phosphorylated form of TDP-43 is disclosed in EuropeanPatent Application No. 2 189 526 A1. Additional anti-TDP-43 antibodiesare disclosed in Zhang et al., Proc Natl Acad Sci USA, 106(18):7607-12(2009) and U.S. Patent Application Pub. No. 20100136573.

The success in generating monoclonal antibodies rests among otherthings, on the efficient and selective fusion of antigen-stimulated Bcells with a murine myeloma cell line followed by selection of stableantibody producing hybrids as originally described by Kóhler andMilstein, Nature 256 (1975), 495-497. However, the therapeutic utilityof murine based antibodies in human is hampered by the human anti-mouseantibody (HAMA) response as a consequence of their non-human origin.Approaches for making human or human-like monoclonal antibodies becameavailable through genetic engineering. However, these methods typicallysuffer from the drawback that they are not suitable for producingantibodies displaying many of the characteristics of antibodies that areendogenously produced by the human immune system during the course of aphysiological human immune response. Furthermore, these geneticallyengineered antibodies may show undesired cross-reactivity with otherproteins and/or the target protein in the context of the biologicallyrelevant native conformation and normal physiological function of thetarget antigen. The resulting side effects upon systemic administrationof the exogenous antibodies may range from for example, undesiredautoimmune disease to anaphylactic reactions. These side effects havebeen reported in so-called “humanized antibodies,” which originally stemfrom non-human organisms, such as mice, as well as so called “fullyhuman antibodies,” in vitro or in xenogeneic mice genetically engineeredto express a repertoire of human antibodies. On the other hand, activeimmunization with pathologically relevant antigens bears theconsiderable risk of patients developing uncontrollable immune responsesagainst these antigens and cross-reactivity with endogenous antigensthat may consequently lead to dangerous autoimmune responses.

Furthermore, the development of assays to detect and monitor levels ofnormal and pathological TDP-43 in plasma, cerebrospinal fluid, and othersamples as biomarkers of FTLD and ALS will provide the ability todiagnose and distinguish TDP-43 proteinopathies from other clinicallysimilar neurodegenerative disorders, such as tauopathies or relatedproteinopathies. In addition, the development of imaging ligands thatenable the detection and/or quantification of TDP-43 neuropathology inliving patients will provide a powerful tool not only for diagnosis, butalso for monitoring the response of patients having a neurodegenerativeTDP-43 proteinopathy to disease-modifying therapies when they becomeavailable.

Thus, there is a need to overcome the above-described limitations and toprovide therapeutic and diagnostic antibodies and other bindingmolecules that specifically recognizes biologically relevantconformations of TDP-43.

SUMMARY OF THE INVENTION

The invention relates to TDP-43 (TAR DNA-binding protein of 43kDa)-specific binding molecules, such as antibodies, includingfragments, derivatives and variants of antibodies that are capable ofspecifically recognizing TDP-43. By “specifically recognizing TDP-43”,“antibody specific to/for TDP-43” and “anti-TDP-43 antibody” is meantspecifically, generally, and collectively, antibodies to TDP-43, ormisfolded or oligomeric or aggregated or posttranslationally modifiedTDP-43. According to one embodiment, antibodies of the invention(including antigen-binding antibody fragments and derivatives)specifically recognize full-length, truncated, or aggregated humanTDP-43. In an additional embodiment, the antibodies recognizefull-length human TDP-43 having the sequence of SEQ ID NO:94 or apeptide consisting of residues 390-414 of the C-terminal sequence of SEQID NO:94 phosphorylated at residues 409 and 410.

In one embodiment, the TDP-43 specific binding molecule is an antibody(including antigen-binding fragments or derivatives thereof) having animmunological binding characteristic of an antibody described herein,such as, an antibody having the variable regions V_(H) and/or V_(L) ofthe amino acid sequence set forth in (SEQ ID NO:1) and (SEQ ID NO:6),(SEQ ID NO:10) and (SEQ ID NO:14), (SEQ ID NO:18) and (SEQ ID NO:22),(SEQ ID NO:26) and (SEQ ID NO:31), (SEQ ID NO:35) and (SEQ ID NO:40),(SEQ ID NO:45) and (SEQ ID NO:49), (SEQ ID NO:53) and (SEQ ID NO:57),(SEQ ID NO:61) and (SEQ ID NO:65), (SEQ ID NO:69) and (SEQ ID NO:73),(SEQ ID NO:77) and (SEQ ID NO:82), (SEQ ID NO:87) and (SEQ ID NO:122),(SEQ ID NO:130) and (SEQ ID NO:134), (SEQ ID NO:138) and (SEQ IDNO:142), (SEQ ID NO:146) and (SEQ ID NO:150), (SEQ ID NO:146) and (SEQID NO:151), (SEQ ID NO:155) and (SEQ ID NO:159), (SEQ ID NO:163) and(SEQ ID NO:167), (SEQ ID NO:171) and (SEQ ID NO:175), (SEQ ID NO:179)and (SEQ ID NO:183), (SEQ ID NO:187) and (SEQ ID NO:191), (SEQ IDNO:195) and (SEQ ID NO:199), (SEQ ID NO:203) and (SEQ ID NO:207), (SEQID NO:211) and (SEQ ID NO:215), (SEQ ID NO:219) and (SEQ ID NO:223),(SEQ ID NO:227) and (SEQ ID NO:213), (SEQ ID NO:235) and (SEQ IDNO:239), (SEQ ID NO:243) and (SEQ ID NO:247), (SEQ ID NO:251) and (SEQID NO:255), (SEQ ID NO:259) and (SEQ ID NO:263), or (SEQ ID NO:267) and(SEQ ID NO:271), respectively.

The invention also relates to compositions comprising an antibody of theinvention (including TDP-43-binding antibody fragments and derivatives)or TDP-43 agonists and cognate molecules, or alternately, antagonists ofthe same and to immunotherapeutic and immunodiagnostic methods usingsuch compositions in the prevention, diagnosis or treatment of a TDP-43proteinopathy, wherein an effective amount of the composition isadministered to a patient in need thereof.

Polynucleotides encoding TDP-43-binding molecules such as, antibodies(including TDP-43 binding antibody fragments or variants), are alsoencompassed by the invention. In some embodiments, the polynucleotideencodes at least a variable region of an immunoglobulin chain of anantibody of the invention. In an additional embodiment, thepolynucleotide encodes at least one complementarity determining region(CDR) of a V_(H) and/or V_(L) variable region as depicted in FIGS. 1 and3. In an additional embodiment, the polynucleotide encodes at least onecomplementarity determining region (CDR) of a V_(H) and/or V_(L)variable region encoded by a polynucleotide sequence as set forth inTable 3. Polynucleotides encoding derivatives or analogs of theabove-encoded TDP-43 binding peptides and the polypeptides encoded bythese polynucleotides are also encompassed by the invention.

Vectors containing polynucleotides encoding TDP-43-binding molecules(e.g., antibodies) of the invention and host cells transformed withthese vectors and/or polynucleotides are also encompassed by theinvention, as are their use for the production of an antibody andequivalent binding molecules which are specific for TDP-43. Means andmethods for the recombinant production of antibodies and other bindingpolypeptides and mimics thereof as well as methods of screening formolecules, e.g., antibodies, that compete with these antibodies or otherbinding proteins for binding with TDP-43 are known in the art. Asdescribed herein, in some embodiments, for example those relating totherapeutic applications in human, the TDP-43 specific binding antibodyof the invention is a human antibody in the sense that application ofsaid antibody is substantially free of a HAMA response otherwiseobserved for chimeric and even humanized antibodies.

In one embodiment, the invention encompasses molecules that specificallybind TDP-43 and the use of these molecules to detect the presence ofTDP-43 in a sample. Accordingly, TDP-43 binding molecules of theinvention, such as, anti-TDP-43 antibodies, can be used to screen humanblood, CSF, and urine for the presence of TDP-43 in samples, forexample, by using ELISA-based or surface adapted assay. The methods andcompositions disclosed herein have applications in diagnosing TDP-43proteinopathies such as, amyotrophic lateral sclerosis (ALS) orfrontotemporal lobar degeneration (FTLD). The methods and compositionsof the invention also have applications in diagnosing presymptomaticdisease and in monitoring disease progression and therapeutic efficacy.According to some embodiments, an antibody specific for TDP-43 (e.g., afull-length antibody or a TDP-43 binding fragment or derivative of anantibody) is contacted with a sample (e.g., blood, cerebrospinal fluid,or brain tissue) to detect, diagnose or monitor frontotemporal lobardegeneration (FTLD) or Amyotrophic lateral sclerosis (ALS). In anotherembodiment, an antibody specific for TDP-43 is contacted with a sampleto detect, diagnose or monitor a disease selected from Alzheimer'sdisease, Parkinson's disease, and Lewy Body disease: In anotherembodiment, an antibody specific for TDP-43 is contacted with a sampleto detect, diagnose or monitor a disease selected from: argyrophilicgrain disease, ALS-Parkinsonism dementia complex of Guam, corticobasaldegeneration, Dementia with Lewy bodies, Huntington's disease, motorneuron disease, frontotemporal lobar degeneration (FTLD), frontotemporaldementia, frontotemporal lobar degeneration with ubiquitin-positiveinclusions, hippocampal sclerosis, inclusion body myopathy, inclusionbody myositis, Parkinson's disease dementia, Parkinson-dementia complexin Kii peninsula, Pick's disease, and Machado-Joseph disease ordementia.

In additional embodiments, the invention provides methods for treatingor preventing a TDP-43 neurologic proteinopathy. According to oneembodiment, the methods of the invention comprise administering aneffective concentration of an antibody specific for TDP-43 (e.g., afull-length antibody or a TDP-43 binding fragment or derivative of anantibody) to a subject. In an additional embodiment, the inventionprovides a method for treating or preventing TDP-43 neurologicproteinopathies. According to some embodiments, an antibody specific forTDP-43 is administered to treat or prevent frontotemporal lobardegeneration (FTLD) or Amyotrophic lateral sclerosis (ALS). In anotherembodiment, an antibody specific for TDP-43 is administered to treat orprevent a neurodegenerative disease selected from Alzheimer's disease,Parkinson's disease, and Lewy Body disease. In another embodiment, anantibody specific for TDP-43 is administered to treat or prevent adisease selected from: argyrophilic grain disease, ALS-Parkinsonismdementia complex of Guam, corticobasal degeneration, Dementia with Lewybodies, Huntington's disease, motor neuron disease, frontotemporal lobardegeneration (FTLD), frontotemporal dementia, frontotemporal lobardegeneration with ubiquitin-positive inclusions, hippocampal sclerosis,inclusion body myopathy, inclusion body myositis, Parkinson's diseasedementia, Parkinson-dementia complex in Kii peninsula, Pick's disease,and Machado-Joseph disease or dementia.

Further embodiments of the invention are apparent from the descriptionand Examples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A: lists the amino acid sequences of the variable region, i.e.heavy chain and kappa/lambda light chain of human antibody NI-205.3F10.Framework (FR), complementarity determining regions (CDRs; underlined),heavy chain joining region (JH), and light chain joining region (JK) or(JL) are indicated. Amino acid positions modified to account forpotential PCR-primer induced cloning artifacts and that have beenmodified to match the corresponding human germ line variable regionsequences are provided in bold.

FIG. 1B: lists the amino acid sequences of the VH and VL of NI-205.51C1.Framework (FR), complementarity determining regions (CDRs; underlined),heavy chain joining region (JH), and light chain joining region (JK) or(JL) are indicated. Amino acid positions modified to account forpotential PCR-primer induced cloning artifacts and that have beenmodified to match the corresponding human germ line variable regionsequences are provided in bold.

FIG. 1C: lists the amino acid sequences of the VH and VL of NI-205.21G2.Framework (FR), complementarity determining regions (CDRs; underlined),heavy chain joining region (JH), and light chain joining region (JK) or(JL) are indicated. Amino acid positions modified to account forpotential PCR-primer induced cloning artifacts and that have beenmodified to match the corresponding human germ line variable regionsequences are provided in bold.

FIG. 1D: lists the amino acid sequences of the VH and VL of NI-205.8A2.Framework (FR), complementarity determining regions (CDRs; underlined),heavy chain joining region (JH), and light chain joining region (JK) or(JL) are indicated. Amino acid positions modified to account forpotential PCR-primer induced cloning artifacts and that have beenmodified to match the corresponding human germ line variable regionsequences are provided in bold.

FIG. 1E: lists the amino acid sequences of the VH and VL ofNI-205.15F12. Framework (FR), complementarity determining regions (CDRs;underlined), heavy chain joining region (JH), and light chain joiningregion (JK) or (JL) are indicated. Amino acid positions modified toaccount for potential PCR-primer induced cloning artifacts and that havebeen modified to match the corresponding human germ line variable regionsequences are provided in bold.

FIG. 1F: lists the amino acid sequences of the VH and VL ofNI-205.113C4. Framework (FR), complementarity determining regions (CDRs;underlined), heavy chain joining region (JH), and light chain joiningregion (JK) or (JL) are indicated. Amino acid positions modified toaccount for potential PCR-primer induced cloning artifacts and that havebeen modified to match the corresponding human germ line variable regionsequences are provided in bold.

FIG. 1G: lists the amino acid sequences of the VH and VL of NI-205.25F3.Framework (FR), complementarity determining regions (CDRs; underlined),heavy chain joining region (JH), and light chain joining region (JK) or(JL) are indicated. Amino acid positions modified to account forpotential PCR-primer induced cloning artifacts and that have beenmodified to match the corresponding human germ line variable regionsequences are provided in bold.

FIG. 1H: lists the amino acid sequences of the VH and VL of NI-205.87E7.Framework (FR), complementarity determining regions (CDRs; underlined),heavy chain joining region (JH), and light chain joining region (JK) or(JL) are indicated. Amino acid positions modified to account forpotential PCR-primer induced cloning artifacts and that have beenmodified to match the corresponding human germ line variable regionsequences are provided in bold.

FIG. 1I: lists the amino acid sequences of the VH and VL of NI-205.21G1.Framework (FR), complementarity determining regions (CDRs; underlined),heavy chain joining region (JH), and light chain joining region (JK) or(JL) are indicated. Amino acid positions modified to account forpotential PCR-primer induced cloning artifacts and that have beenmodified to match the corresponding human germ line variable regionsequences are provided in bold.

FIG. 1J: lists the amino acid sequences of the VH and VL of NI-205.68G5.Framework (FR), complementarity determining regions (CDRs; underlined),heavy chain joining region (JH), and light chain joining region (JK) or(JL) are indicated. Amino acid positions modified to account forpotential PCR-primer induced cloning artifacts and that have beenmodified to match the corresponding human germ line variable regionsequences are provided in bold.

FIG. 1K: lists the amino acid sequences of the VH and VL of NI-205.20A1.Framework (FR), complementarity determining regions (CDRs; underlined),heavy chain joining region (JH), and light chain joining region (JK) or(JL) are indicated. Amino acid positions modified to account forpotential PCR-primer induced cloning artifacts and that have beenmodified to match the corresponding human germ line variable regionsequences are provided in bold.

FIG. 2A: is a bar graph depicting the binding of human TDP-43 antibody,205.51C1, to amino terminal His-tagged fragments of TDP-43 consisting ofamino acid residues 2-106 (domain I (SEQ ID NO:117)), 99-204 (domain II(SEQ ID NO:118)), 183-273 (domain III (SEQ ID NO:119)), 258-414 (domainIV (SEQ ID NO:120)), or 2-414 (full length (SEQ ID NO:121)).

FIG. 2B: is a bar graph depicting the binding of human TDP-43 antibody,205.21G2, to amino terminal His-tagged fragments of TDP-43 consisting ofamino acid residues 2-106 (domain I (SEQ ID NO:117)), 99-204 (domain II(SEQ ID NO:118)), 183-273 (domain III (SEQ ID NO:119)), 258-414 (domainIV (SEQ ID NO:120)), or 2-414 (full length (SEQ ID NO:121)).

FIG. 2C: is a bar graph depicting the binding of human TDP-43 antibody,205.3F10, to amino terminal His-tagged fragments of TDP-43 consisting ofamino acid residues 2-106 (domain I (SEQ ID NO:117)), 99-204 (domain II(SEQ ID NO:118)), 183-273 (domain III (SEQ ID NO:119)), 258-414 (domainIV (SEQ ID NO:120)), or 2-414 (full length (SEQ ID NO:121)).

FIG. 2D: is a bar graph depicting the binding of human TDP-43 antibody,205.8A2, to amino terminal His-tagged fragments of TDP-43 consisting ofamino acid residues 2-106 (domain I (SEQ ID NO:117)), 99-204 (domain II(SEQ ID NO:118)), 183-273 (domain III (SEQ ID NO:119)), 258-414 (domainIV (SEQ ID NO:120)), or 2-414 (full length (SEQ ID NO:121)).

FIG. 2E: is a bar graph depicting the binding of human TDP-43 antibody,205.15F12, to amino terminal His-tagged fragments of TDP-43 consistingof amino acid residues 2-106 (domain I (SEQ ID NO:117)), 99-204 (domainII (SEQ ID NO:118)), 183-273 (domain III (SEQ ID NO:119)), 258-414(domain IV (SEQ ID NO:120)), or 2-414 (full length (SEQ ID NO:121)).

FIG. 2F: is a bar graph depicting the binding of human TDP-43 antibody,205.113C4, to amino terminal His-tagged fragments of TDP-43 consistingof amino acid residues 2-106 (domain I (SEQ ID NO:117)), 99-204 (domainII (SEQ ID NO:118)), 183-273 (domain III (SEQ ID NO:119)), 258-414(domain IV (SEQ ID NO:120)), or 2-414 (full length (SEQ ID NO:121)).

FIG. 2G: is a bar graph depicting the binding of human TDP-43 antibody,205.25F3, to amino terminal His-tagged fragments of TDP-43 consisting ofamino acid residues 2-106 (domain I (SEQ ID NO:117)), 99-204 (domain II(SEQ ID NO:118)), 183-273 (domain III (SEQ ID NO:119)), 258-414 (domainIV (SEQ ID NO:120)), or 2-414 (full length (SEQ ID NO:121)).

FIG. 2H: is a bar graph depicting the binding of human TDP-43 antibody,205.87E7, to amino terminal His-tagged fragments of TDP-43 consisting ofamino acid residues 2-106 (domain I (SEQ ID NO:117)), 99-204 (domain II(SEQ ID NO:118)), 183-273 (domain III (SEQ ID NO:119)), 258-414 (domainIV (SEQ ID NO:120)), or 2-414 (full length (SEQ ID NO:121)).

FIG. 2I: is a bar graph depicting the binding of human TDP-43 antibody,205.21G1, to amino terminal His-tagged fragments of TDP-43 consisting ofamino acid residues 2-106 (domain I (SEQ ID NO:117)), 99-204 (domain II(SEQ ID NO:118)), 183-273 (domain III (SEQ ID NO:119)), 258-414 (domainIV (SEQ ID NO:120)), or 2-414 (full length (SEQ ID NO:121)).

FIG. 2J: is a bar graph depicting the binding of Abnova 23435, Abcam50930, Abcam 82695, and anti-human Fcγ, to amino terminal His-taggedfragments of TDP-43 consisting of amino acid residues 2-106 (domain I(SEQ ID NO:117)), 99-204 (domain II (SEQ ID NO:118)), 183-273 (domainIII (SEQ ID NO:119)), 258-414 (domain IV (SEQ ID NO:120)), or 2-414(full length (SEQ ID NO:121)).

FIG. 3A: lists the amino acid sequences of the variable region, i.e.heavy chain (VH) and kappa (VK)/lambda (VL) light chain of humananti-TDP-43 antibody, NI205.41D1. Complementarity determining regions(CDRs) are underlined.

FIG. 3B: lists the amino acid sequences of the VH and VL of humananti-TDP-43 antibody, NI205.29E11. Complementarity determining regions(CDRs) are underlined.

FIG. 3C: lists the amino acid sequences of the VH and VL of humananti-TDP-43 antibody, NI205.9E12. Complementarity determining regions(CDRs) are underlined.

FIG. 3D: lists the amino acid sequences of the VH and VL of humananti-TDP-43 antibody, NI205.98H6. Complementarity determining regions(CDRs) are underlined.

FIG. 3E: lists the amino acid sequences of the VH and VL of humananti-TDP-43 antibody, NI205.10D3. Complementarity determining regions(CDRs) are underlined.

FIG. 3F: lists the amino acid sequences of the VH and VL of humananti-TDP-43 antibody, NI205.44B2. Complementarity determining regions(CDRs) are underlined.

FIG. 3G: lists the amino acid sequences of the VH and VL of humananti-TDP-43 antibody, NI205.38H2. Complementarity determining regions(CDRs) are underlined.

FIG. 3H: lists the amino acid sequences of the VH and VL of humananti-TDP-43 antibody, NI205.36D5. Complementarity determining regions(CDRs) are underlined.

FIG. 3I: lists the amino acid sequences of the VH and VL of humananti-TDP-43 antibody, NI205.58E11. Complementarity determining regions(CDRs) are underlined.

FIG. 3J: lists the amino acid sequences of the VH and VL of humananti-TDP-43 antibody, NI205.14H5. Complementarity determining regions(CDRs) are underlined.

FIG. 3K: lists the amino acid sequences of the VH and VL of humananti-TDP-43 antibody, NI205.31D2. Complementarity determining regions(CDRs) are underlined.

FIG. 3L: lists the amino acid sequences of the VH and VL of humananti-TDP-43 antibody, NI205.8F8. Complementarity determining regions(CDRs) are underlined.

FIG. 3M: lists the amino acid sequences of the VH and VL of humananti-TDP-43 antibody, NI205.31C11. Complementarity determining regions(CDRs) are underlined.

FIG. 3N: lists the amino acid sequences of the VH and VL of humananti-TDP-43 antibody, NI205.8C10. Complementarity determining regions(CDRs) are underlined.

FIG. 3O: lists the amino acid sequences of the VH and VL of humananti-TDP-43 antibody, NI205.10H7. Complementarity determining regions(CDRs) are underlined.

FIG. 3P: lists the amino acid sequences of the VH and VL of humananti-TDP-43 antibody, NI205.1A9. Complementarity determining regions(CDRs) are underlined.

FIG. 3Q: lists the amino acid sequences of the VH and VL of humananti-TDP-43 antibody, NI205.14W3. Complementarity determining regions(CDRs) are underlined.

FIG. 3R: lists the amino acid sequences of the VH and VL of humananti-TDP-43 antibody, NI205.19G5. Complementarity determining regions(CDRs) are underlined.

FIG. 4A: is a graph of the half maximal effective concentration (EC₅₀)of human-derived TDP-43 antibody, 41D1, by direct ELISA to recombinantfull length TDP-43 (●), Escherichia coli extract (▴) and BSA (▪).

FIG. 4B: is a graph of the EC₅₀ of human-derived TDP-43 antibody, 21G1,by direct ELISA to recombinant full length TDP-43 (●), Escherichia coliextract (▴) and BSA (▪).

FIG. 4C: is a graph of the EC₅₀ of human-derived TDP-43 antibody, 31D2,by direct ELISA to recombinant full length TDP-43 (●), Escherichia coliextract (▴) and BSA (▪).

FIG. 4D: is a graph of the EC₅₀ of human-derived TDP-43 antibody, 8F8,by direct ELISA to recombinant full length TDP-43 (●), Escherichia coliextract (▴) and BSA (▪).

FIG. 4E: is a graph of the EC₅₀ of human-derived TDP-43 antibody, 41D1,by direct ELISA to a synthetic peptide covering residues 390 to 414 ofthe C-terminal domain of TDP-43 with phosphorylation modification atresidues 409/410 (●) and BSA (▪).

FIG. 4F: is a graph of the EC₅₀ of human-derived TDP-43 antibody, 21G1,by direct ELISA to a synthetic peptide covering residues 390 to 414 ofthe C-terminal domain of TDP-43 with phosphorylation modification atresidues 409/410 (●) and BSA (▪).

FIG. 4G: is a graph of the EC₅₀ of human-derived TDP-43 antibody, 31D2,by direct ELISA to a synthetic peptide covering residues 390 to 414 ofthe C-terminal domain of TDP-43 with phosphorylation modification atresidues 409/410 (●) and BSA (▪).

FIG. 4H: is a graph of the EC₅₀ of human-derived TDP-43 antibody, 8F8,by direct ELISA to a synthetic peptide covering residues 390 to 414 ofthe C-terminal domain of TDP-43 with phosphorylation modification atresidues 409/410 (●) and BSA (▪).

FIG. 5A: is a bar graph depicting the binding specificity of thehuman-derived antibody, 41D1, to TDP-43 domains comprising amino acids2-106 (domain I), 99-204 (domain II), 183-273 (domain III), 258-414(domain IV) and 2-414 (full length) of TDP-43 as determined by directELISA.

FIG. 5B: is a bar graph depicting the binding specificity of thehuman-derived antibody, 1A9, to TDP-43 domains comprising amino acids2-106 (domain I), 99-204 (domain II), 183-273 (domain III), 258-414(domain IV) and 2-414 (full length) of TDP-43 as determined by directELISA.

FIG. 5C: is a bar graph depicting the binding specificity of thehuman-derived antibody, 14W3, to TDP-43 domains comprising amino acids2-106 (domain I), 99-204 (domain II), 183-273 (domain III), 258-414(domain IV) and 2-414 (full length) of TDP-43 as determined by directELISA.

FIG. 5D: is a bar graph depicting the binding specificity of thehuman-derived antibody, 98H6, to TDP-43 domains comprising amino acids2-106 (domain I), 99-204 (domain II), 183-273 (domain III), 258-414(domain IV) and 2-414 (full length) of TDP-43 as determined by directELISA.

FIG. 5E: is a bar graph depicting the binding specificity of thehuman-derived antibody, 44B2, to TDP-43 domains comprising amino acids2-106 (domain I), 99-204 (domain II), 183-273 (domain III), 258-414(domain IV) and 2-414 (full length) of TDP-43 as determined by directELISA.

FIG. 5F: is a bar graph depicting the binding specificity of thehuman-derived antibody, 9E12A, to TDP-43 domains comprising amino acids2-106 (domain I), 99-204 (domain II), 183-273 (domain III), 258-414(domain IV) and 2-414 (full length) of TDP-43 as determined by directELISA.

FIG. 5G: is a bar graph depicting the binding specificity of thehuman-derived antibody, 10D3, to TDP-43 domains comprising amino acids2-106 (domain I), 99-204 (domain II), 183-273 (domain III), 258-414(domain IV) and 2-414 (full length) of TDP-43 as determined by directELISA.

FIG. 5H: is a bar graph depicting the binding specificity of thehuman-derived antibody, 38H2, to TDP-43 domains comprising amino acids2-106 (domain I), 99-204 (domain II), 183-273 (domain III), 258-414(domain IV) and 2-414 (full length) of TDP-43 as determined by directELISA.

FIG. 5I: is a bar graph depicting the binding specificity of thehuman-derived antibody, 29E11, to TDP-43 domains comprising amino acids2-106 (domain I), 99-204 (domain II), 183-273 (domain III), 258-414(domain IV) and 2-414 (full length) of TDP-43 as determined by directELISA.

FIG. 5J: is a bar graph depicting the binding specificity of thehuman-derived antibody, 9E12D, to TDP-43 domains comprising amino acids2-106 (domain I), 99-204 (domain II), 183-273 (domain III), 258-414(domain IV) and 2-414 (full length) of TDP-43 as determined by directELISA.

FIG. 5K: is a bar graph depicting the binding specificity of thehuman-derived antibody, 31C11, to TDP-43 domains comprising amino acids2-106 (domain I), 99-204 (domain II), 183-273 (domain III), 258-414(domain IV) and 2-414 (full length) of TDP-43 as determined by directELISA.

FIG. 5L: is a bar graph depicting the binding specificity of thehuman-derived antibody, 10H7, to TDP-43 domains comprising amino acids2-106 (domain I), 99-204 (domain II), 183-273 (domain III), 258-414(domain IV) and 2-414 (full length) of TDP-43 as determined by directELISA.

FIG. 5M: is a bar graph depicting the binding specificity of thehuman-derived antibody, 8C10, to TDP-43 domains comprising amino acids2-106 (domain I), 99-204 (domain II), 183-273 (domain III), 258-414(domain IV) and 2-414 (full length) of TDP-43 as determined by directELISA.

FIG. 6A: is an immunoblot showing the binding of the human-derivedantibody, 41D1, to TDP-43 domains comprising amino acids 2-414 (fulllength), 2-106 (domain I), 99-204 (domain II), 183-273 (domain III), and258-414 (domain IV) of TDP-43 as determined by Western Blot analysis.

FIG. 6B: is an immunoblot showing the binding of the human-derivedantibody, 51C1, to TDP-43 domains comprising amino acids 2-414 (fulllength), 2-106 (domain I), 99-204 (domain II), 183-273 (domain III), and258-414 (domain IV) of TDP-43 as determined by Western Blot analysis.

FIG. 6C: is an immunoblot showing the binding of the human-derivedantibody, 21G2, to TDP-43 domains comprising amino acids 2-414 (fulllength), 2-106 (domain I), 99-204 (domain II), 183-273 (domain III), and258-414 (domain IV) of TDP-43 as determined by Western Blot analysis.

FIG. 6D: is an immunoblot showing the binding of the human-derivedantibody, 1A9, to TDP-43 domains comprising amino acids 2-414 (fulllength), 2-106 (domain I), 99-204 (domain II), 183-273 (domain III), and258-414 (domain IV) of TDP-43 as determined by Western Blot analysis.

FIG. 6E: is an immunoblot showing the binding of the human-derivedantibody, 3F10, to TDP-43 domains comprising amino acids 2-414 (fulllength), 2-106 (domain I), 99-204 (domain II), 183-273 (domain III), and258-414 (domain IV) of TDP-43 as determined by Western Blot analysis.

FIG. 6F: is an immunoblot showing the binding of the human-derivedantibody, 14W3, to TDP-43 domains comprising amino acids 2-414 (fulllength), 2-106 (domain I), 99-204 (domain II), 183-273 (domain III), and258-414 (domain IV) of TDP-43 as determined by Western Blot analysis.

FIG. 6G: is an immunoblot showing the binding of the human-derivedantibody, 98H6, to TDP-43 domains comprising amino acids 2-414 (fulllength), 2-106 (domain I), 99-204 (domain II), 183-273 (domain III), and258-414 (domain IV) of TDP-43 as determined by Western Blot analysis.

FIG. 6H: is an immunoblot showing the binding of the human-derivedantibody, 8A2, to TDP-43 domains comprising amino acids 2-414 (fulllength), 2-106 (domain I), 99-204 (domain II), 183-273 (domain III), and258-414 (domain IV) of TDP-43 as determined by Western Blot analysis.

FIG. 6I: is an immunoblot showing the binding of the human-derivedantibody, 15F12, to TDP-43 domains comprising amino acids 2-414 (fulllength), 2-106 (domain I), 99-204 (domain II), 183-273 (domain III), and258-414 (domain IV) of TDP-43 as determined by Western Blot analysis.

FIG. 6J: is an immunoblot showing the binding of the human-derivedantibody, 10D3, to TDP-43 domains comprising amino acids 2-414 (fulllength), 2-106 (domain I), 99-204 (domain II), 183-273 (domain III), and258-414 (domain IV) of TDP-43 as determined by Western Blot analysis.

FIG. 6K: is an immunoblot showing the binding of the human-derivedantibody, 31C11, to TDP-43 domains comprising amino acids 2-414 (fulllength), 2-106 (domain I), 99-204 (domain II), 183-273 (domain III), and258-414 (domain IV) of TDP-43 as determined by Western Blot analysis.

FIG. 6L: is an immunoblot showing the binding of the human-derivedantibody, 10H7, to TDP-43 domains comprising amino acids 2-414 (fulllength), 2-106 (domain I), 99-204 (domain II), 183-273 (domain III), and258-414 (domain IV) of TDP-43 as determined by Western Blot analysis.

FIG. 6M: is an immunoblot showing the binding of the human-derivedantibody, 21G1, to TDP-43 domains comprising amino acids 2-414 (fulllength), 2-106 (domain I), 99-204 (domain II), 183-273 (domain III), and258-414 (domain IV) of TDP-43 as determined by Western Blot analysis.

FIG. 6N: is an immunoblot showing the binding of the human-derivedantibody, 68G5, to TDP-43 domains comprising amino acids 2-414 (fulllength), 2-106 (domain I), 99-204 (domain II), 183-273 (domain III), and258-414 (domain IV) of TDP-43 as determined by Western Blot analysis.

FIG. 6O: is an immunoblot showing the binding of the human-derivedantibody, 20A1, to TDP-43 domains comprising amino acids 2-414 (fulllength), 2-106 (domain I), 99-204 (domain II), 183-273 (domain III), and258-414 (domain IV) of TDP-43 as determined by Western Blot analysis.

FIG. 6P: is an immunoblot showing the binding of the antibody, Abnova23435, to TDP-43 domains comprising amino acids 2-414 (full length),2-106 (domain I), 99-204 (domain II), 183-273 (domain III), and 258-414(domain IV) of TDP-43 as determined by Western Blot analysis.

FIG. 6Q: is an immunoblot showing the binding of the anti-human aIgG Fcγantibody to TDP-43 domains comprising amino acids 2-414 (full length),2-106 (domain I), 99-204 (domain II), 183-273 (domain III), and 258-414(domain IV) of TDP-43 as determined by Western Blot analysis.

FIG. 7A: is a bar graph showing NI-205.41D1 antibody binding to fulllength TDP-43 and TDP-43 fragments comprising amino acid residues258-414, 258-384, 258-375, 258-362, 258-353, 258-319, 317-414 and340-414.

FIG. 7B: is a bar graph showing NI-205.41D1 and 12892-1-AP antibodybinding to full length TDP-43, TDP-43 fragment comprising amino acidresidues 258-414, and to a mutant TDP-43 polypeptide fragment comprisingamino acid residues 258-414 and the A to G substitution at residue 321,M to G substitution at residue 322, and M to G substitution at residue323 (TDP-43 258-414 AMM321GGG).

FIG. 7C: is a bar graph showing NI-205.41D1 antibody binding to TDP-43fragments comprising amino acid residues 316-353, 316-343, and 316-333.

FIG. 8A: is an image of a Coomassie stained SDS-PAGE gel of purifiedTDP-43 forms in the presence or absence of chaotrope KSCN. Lane 1 to 4,respectively: Molecular Weight standards, 6×His-TDP-43 (1-414),6×His-SUMO-TDP-43 (1-414), 6His-SUMO-TDP-43 (220-414), all purified inthe presence of 1.5M KSCN, and lane 5 and 6: 6×His-SUMO-TDP-43 (101-265)purified in the presence of KCl (lane 5) or KSCN (lane 6)

FIG. 8B: is a schematic diagram of the domain arrangement of purifiedproteins illustrating location of RNA binding domains (RRM1 and RRM2) aswell as tags (6His and SUMO).

FIG. 8C: provides graphical depictions of analytical ultracentrifugationsedimentation coefficient distributions for purified 6His-SUMO and 6Histagged full-length TDP-43. Sedimentation coefficients and calculatedmolecular weights are shown above peaks.

FIG. 9A: is a graphical depiction of full-length TDP-43 binding tospecific ((UG)₆) or control ((UU)₆) RNA in buffer containing 40 mMHEPES, 0.5 M KCl, 0.4 M Arginine, pH 7.4.

FIG. 9B: is a graphical depiction of RNA binding by TDP-43 (101-265) inthe presence of a physiological buffer containing 20 mM HEPES, 80 mMpotassium glutamate, 4 mM magnesium acetate, 5% glycerol, pH 7.5, 2 mMDTT, to determine K_(d).

FIG. 9C: is a graphical depiction of RNA binding by TDP-43 (101-265) inthe presence of a physiological buffer containing 20 mM HEPES, 80 mMpotassium glutamate, 4 mM magnesium acetate, 5% glycerol, pH 7.5, 2 mMDTT, to determine stoichiometry when purified in non-chaotropicconditions.

FIG. 9D: is a graphical depiction of RNA binding by TDP-43 (101-265) inthe presence of a physiological buffer containing 20 mM HEPES, 80 mMpotassium glutamate, 4 mM magnesium acetate, 5% glycerol, pH 7.5, 2 mMDTT, to determine stoichiometry when purified in mild chaotropicconditions.

FIG. 10A: is a graphical depiction of titration curves for humanNI-205.41D1, NI-205.21G1, NI-205.51C1, NI-205.21G2, and NI-205.3F10antibody binding to folded full-length TDP-43.

FIG. 10B: is a graphical depiction of titration curves for humanNI-205.41D1, NI-205.21G1, NI-205.51C1, NI-205.21G2, and NI-205.3F10antibody binding to TDP-43 fragment comprising residues 220-414 in acapture ELISA assay.

FIG. 11A: is an image of immunohistochemical staining of human FTLD-Uhippocampus tissue with antibodies 2E2-D3.

FIG. 11B: is an image of immunohistochemical staining of human FTLD-Uhippocampus tissue with antibodies 2E2-D3.

FIG. 11C: is an image of immunohistochemical staining of human FTLD-Uhippocampus tissue with antibodies 2E2-D3.

FIG. 11D: is an image of immunohistochemical staining of human FTLD-Uhippocampus tissue with control antibody p403/p404.

FIG. 11E: is an image of immunohistochemical staining of human FTLD-Uhippocampus tissue with control antibody p403/p404.

FIG. 11F: is an image of immunohistochemical staining of human FTLD-Uhippocampus tissue with control antibody p403/p404.

FIG. 11G: is an image of immunohistochemical staining of human FTLD-Uhippocampus tissue with control antibody p409/p410.

FIG. 11H: is an image of immunohistochemical staining of human FTLD-Uhippocampus tissue with control antibody p409/p410.

FIG. 11I: is an image of immunohistochemical staining of human FTLD-Uhippocampus tissue with control antibody p409/p410.

FIG. 11J: is an image of immunohistochemical staining of human FTLD-Uhippocampus tissue with antibody NI-205.10D3.

FIG. 11K: is an image of immunohistochemical staining of human FTLD-Uhippocampus tissue with antibody NI-205.8C10.

FIG. 11L: is an image of immunohistochemical staining of human FTLD-Uhippocampus tissue with antibody NI-205.15F12.

FIG. 11M: is an image of immunohistochemical staining of human FTLD-Uhippocampus tissue with antibody NI-205.8A2.

FIG. 11N: is an image of immunohistochemical staining of human FTLD-Uhippocampus tissue with antibody NI-205.3F10.

FIG. 11O: is an image of immunohistochemical staining of human FTLD-Uhippocampus tissue with antibody NI-205.21G2.

FIG. 11P: is an image of immunohistochemical staining of human FTLD-Uhippocampus tissue with antibody NI-205.8F8.

FIG. 11Q: is an image of immunohistochemical staining of human FTLD-Uhippocampus tissue with antibody NI-205.31C11.

FIG. 11R: is an image of immunohistochemical staining of human FTLD-Uhippocampus tissue with antibody NI-205.36D5.

FIG. 11S: is an image of immunohistochemical staining of human FTLD-Uhippocampus tissue with antibody NI-205.31D2.

FIG. 11T: is an image of immunohistochemical staining of human FTLD-Uhippocampus tissue with antibody NI-205.10H7.

FIG. 11U: is an image of immunohistochemical staining of human FTLD-Uhippocampus tissue with antibody NI-205.14H5.

FIG. 11V: is an image of immunohistochemical staining of human FTLD-Uhippocampus tissue with antibody NI-205.68G5.

FIG. 11W: is an image of immunohistochemical staining of human FTLD-Uhippocampus tissue with antibody NI-205.14W3.

FIG. 11X: is an image of immunohistochemical staining of human FTLD-Uhippocampus tissue with antibody NI-205.21G1.

FIG. 11Y: is an image of immunohistochemical staining of human FTLD-Uhippocampus tissue with antibody NI-205.41D1.

FIG. 11Z: is an image of immunohistochemical staining of controlhippocampus tissue with NI-205.41D1.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

As used herein, the phrase “neurodegenerative diseases” refers topresence of abnormal protein accumulation, cellular localization, orprotein folding in the brain, spinal cord, or other neural tissue andare caused by the death or functional impairment of neurons. In somecases of neurodegenerative diseases, genetically defined abnormalitiescontribute to the development of the disease. Neurodegenerative diseasesinclude for example, cerebral degenerative disease (e.g., Alzheimer'sdisease, Parkinson's disease, progressive supranuclear palsy, andHuntington's disease) and spinal degenerative diseases/motor neurondegenerative diseases e.g., amyotrophic lateral sclerosis and spinalmuscular atrophy; see, e.g., Forman et al. Nat. Med. 10 (2004), 1055-63.

“TDP-43 proteinopathy” relates to the nervous system diseases, inparticular to neurodegenerative diseases and are known as a heterologousgroup of disorders linked by the TAR (Transactivationresponsive)-DNA-binding protein of 43 kDa. TDP-43 proteinopathies arecharacterized by the fact that TDP-43 is a disease protein thatmechanistically links frontotemporal lobar degeneration withubiquitin-positive inclusions (FTLD-U) with and without motor neurondisease to amyotrophic lateral sclerosis (ALS) argyrophilic graindisease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS),ALS-Parkinsonism dementia complex of Guam, corticobasal degeneration,Dementia with Lewy bodies, Huntington's disease, Lewy body disease,motor neuron disease, frontotemporal lobar degeneration (FTLD),frontotemporal dementia, frontotemporal lobar degeneration withubiquitin-positive inclusions, hippocampal sclerosis, inclusion bodymyopathy, inclusion body myositis, Parkinson's disease, Parkinson'sdisease dementia, Parkinson-dementia complex in Kii peninsula, Pick'sdisease, Machado-Joseph disease and the like; see e.g., Lagier-Tourenneet al., Hum. Mol. Gen. 19 (2010), R46-64, which is herein incorporatedby reference in its entirety.

Under normal physiological conditions, TDP-43 predominantly localizes tothe nucleus. However, a substantial loss of nuclear TDP-43 is observedin neurons bearing aberrant cytoplasmic TDP-43 inclusions. TDP-43exhibits a disease-specific biochemical signature; pathologicallyaltered TDP-43. TDP-43 proteinopathies are distinct from most otherneurodegenerative disorders in which protein misfolding leads to brainamyloidosis, as pathologic TDP-43 forms neuronal and glial inclusionslacking the features of brain amyloid deposits; see e.g., Neumann etal., Arch Neurol. 64 (2007), 1388-1394, herein incorporated by referencein its entirety.

As used herein, the term “pathologic TDP-43” refers to extracellular,cytoplasmic, neuritic, and nuclear inclusions, is also referred to as a“TDP-43 inclusion body” wherein the protein forms fibril-like clumps.Specifically, pathologic TDP-43 has been found to behyperphosphorylated, ubiquitinated, and N-terminally truncated, therebygenerating abnormal species of TDP-43 that migrate with a highermolecular mass at approximately 45 kDa, as well as a smear ofhigh-molecular-mass proteins and C-terminal fragments of approximately25 kDa; see, e.g., Neumann et al., Science 314 (2006), 130-133 and Araiet al., Biochem. Biophys. Res. Commun. 351 (2006), 602-611, each ofwhich is herein incorporated by reference in its entirety. Additionally,TDP-43 has been found to exhibit multiple phosphorylation sites incarboxyl-terminal regions of deposited TDP-43 and it is suggested thatphosphorylation leads to increased oligomerization and fibrillization ofTDP-43; see, for example, Hasegawa et al., Annals of Neurology 64(2008), 60-70.

TDP-43 inclusion body formation is accompanied by change in thesubcellular distribution of TDP-43 with complete lack of normal diffusenuclear TDP-43 staining in inclusion-bearing cells. The presence andextent of this pathologic signature in affected cortical gray and whitematter, as well as the spinal cord, roughly correspond with the densityof TDP-43-positive inclusions; see, e.g., Neumann et al., J. Neuropath.Exp. Neurol. 66 (2007), 177-183. The composition of the ubiquitinatedinclusions (UBIs) in FTLD-U is characterized by a relative lowabundance, uneven distribution of UBIs among different FTLD-U cases, andthe non-amyloidogenic nature of them. Thus, TDP-43 is a specific andsensitive marker to detect the characteristic ubiquitin-immunoreactivelesions in FTLD-U, including neuronal cytoplasmic inclusions (NCIs),dystrophic neurites, and neuronal intranuclear inclusions (NIIs).

As used herein, the terms “TARDBP,” “Transactivation responsive-DNAbinding protein of 43 kDa”, “Transactive responsive-DNA binding proteinof 43 kDa”, “TAR-DNA binding protein of 43kDA” and “TDP-43” are usedinterchangeably to refer to the native form of TDP-43. The term “TDP-43”is also used to refer collectively to all types and forms of TDP-43.“TDP-43” is also used to generally identify other conformers of TDP-43,including for example, phosphorylated forms of TDP-43 andubiquitin-associated aggregates or aggregates of TDP-43.

The amino acid sequence of human TDP-43 is known in the art; see, e.g.,Strausberg et al., TARDBP protein (Homo sapiens) GenBank Pubmed:AAH71657 version GI:47939520 herein incorporated by reference in itsentirety. According to one embodiment, the amino acid sequence of nativehuman TDP-43 is:

(SEQ ID NO: 94) MSEYIRVTEDENDEPIEIPSEDDGTVLLSTVTAQFPGACGLRYRNPVSQCMRGVRLVEGILHAPDAGWGNLVYVVNYPKDNKRKMDETDASSAVKVKRAVQKTSDLIVLGLPWKTTEQDLKEYFSTFGEVLMVQVKKDLKTGHSKGFGFVRFTEYETQVKVMSQRHMIDGRWCDCKLPNSKQSQDEPLRSRKVFVGRCTEDMTEDELREFFSQYGDVMDVFIPKPFRAFAFVTFADDQIAQSLCGEDLIIKGISVHISNAEPKHNSNRQLERSGRFGGNPGGFGNQGGFGNSRGGGAGLGNNQGSNMGGGMNFGAFSINPAMMAAAQAALQSSWGMMGMLASQQNQSGPSGNNQNQGNMQREPNQAFGSGNNSYSGSNSGAAIGWGSASNAGSGSGFNGGFGSSMDSKSSGWGM

As used herein, the term “antibody” or “antibodies” is meant to refer tocomplete, intact antibodies, and Fab, Fab′, F(ab)2, and other antibodyfragments. Complete, intact antibodies include, but are not limited to,monoclonal antibodies such as murine monoclonal antibodies, polyclonalantibodies, chimeric antibodies, human antibodies, and humanizedantibodies. Various forms of antibodies can be produced using standardrecombinant DNA techniques (Winter and Milstein, Nature 349 (1991),293-99. For example, “chimeric” antibodies can be constructed, in whichthe antigen binding domain from an animal antibody is linked to a humanconstant domain (an antibody derived initially from a nonhuman mammal inwhich recombinant DNA technology has been used to replace all or part ofthe hinge and constant regions of the heavy chain and/or the constantregion of the light chain, with corresponding regions from a humanimmunoglobulin light chain or heavy chain) (see, e.g., U.S. Pat. No.4,816,567; Morrison et al., Proc. Natl. Acad. Sci. 81 (1984), 6851-6855.Chimeric antibodies reduce the immunogenic responses elicited by animalantibodies when used in human clinical treatments.

In addition, recombinant “humanized” antibodies can be synthesized.Humanized antibodies are antibodies initially derived from a nonhumanmammal in which recombinant DNA technology has been used to substitutesome or all of the amino acids not required for antigen binding withamino acids from corresponding regions of a human immunoglobulin lightor heavy chain. That is, they are chimeras comprising mostly humanimmunoglobulin sequences into which the regions responsible for specificantigen-binding have been inserted (see, e.g., International PatentApplication Publication No. WO 94/04679). Animals are immunized with thedesired antigen, the corresponding antibodies are isolated and theportions of the variable region sequences responsible for specificantigen binding are removed. The animal-derived antigen binding regionsare then cloned into the appropriate position of the human antibodygenes in which the antigen binding regions have been deleted. Humanizedantibodies minimize the use of heterologous (inter-species) sequences inantibodies for use in human therapies, and are less likely to elicitunwanted immune responses. Primatized antibodies can be producedsimilarly.

Additional embodiments of the invention relate to human antibodies aswell as the uses of these human antibodies. In one embodiment, the humanantibodies are derived from human B cells or other immune cells and aregenerally referred to herein as “completely human antibodies.” Thus, theinvention encompasses the immortalized human B memory lymphocyte and Bcell, respectively, that produces the antibody having the distinct andunique characteristics as defined below. Alternatively, human antibodiescan be produced in nonhuman animals, such as transgenic animalsharboring one or more human immunoglobulin transgenes. Such animals canbe used as a source for splenocytes for producing hybridomas, as isdescribed in U.S. Pat. No. 5,569,825.

The antigen-binding fragments of antibodies of the invention can be asingle chain Fv fragment, an F(ab′) fragment, an F(ab) fragment, and anF(ab′)2 fragment. In additional embodiments, the antigen-bindingfragment is a TDP-43-binding fragment that recognizes TDP-43 by one ormore antibody variable domains, or fragments or variants thereto, suchas, one or more CDRs or a derivative) of one or more CDS. In a specificembodiment, infra, the antibody or fragment thereof is a human IgGisotype antibody. Alternatively, the antibody is a chimeric human-murineor murinized antibody, the latter being particularly useful fordiagnostic methods and studies in animals.

Antibody fragments, univalent antibodies, and single domain antibodiescan also be used in the methods and compositions of the invention.Univalent antibodies comprise a heavy chain/light chain dimer bound tothe Fc (or stem) region of a second heavy chain. “Fab region” refers tothose portions of the chains which are roughly equivalent, or analogous,to the sequences which comprise the Y branch portions of the heavy chainand to the light chain in its entirety, and which collectively (inaggregates) have been shown to exhibit antibody activity. A Fab proteinincludes aggregates of one heavy and one light chain (commonly known asFab′), as well as tetramers which correspond to the two branch segmentsof the antibody Y, (commonly known as F(ab)2), whether any of the aboveare covalently or non-covalently aggregated, so long as the aggregationis capable of specifically reacting with a particular antigen or antigenfamily.

The TDP-43 antibodies of the invention specifically bind TDP-43,epitopes of TDP-43, and to various conformations of TDP-43 and epitopesthereof. For example, disclosed herein are antibodies that specificallybind native TDP-43, full-length and truncated TDP-43, and pathologicTDP-43. As used herein, reference to an antibody that “specificallybinds”, “selectively binds”, or “preferentially binds” or even moregenerally “binds TDP-43” or “TDP-43 binding”, refers to an antibody thatbinds TDP-43 preferentially over other distinct proteins. As usedherein, an antibody that “specifically binds” or “selectively binds” anTDP-43 conformer does not bind at least one other TDP-43 conformer. Forexample, disclosed herein are antibodies that selectively bindfull-length TDP-43 as well as those that selectively bind cytoplasmicTDP-43 over nuclear TDP-43.

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “an antibody,” is understood to representone or more antibodies. As such, the terms “a” (or “an”), “one or more,”and “at least one” can be used interchangeably herein.

As used herein, the term “polypeptide” is used interchangeably with theterm “polypeptide” and encompasses a singular polypeptide/protein aswell as plural polypeptides/proteins and refers to any chain or chainsof two or more amino acids, and does not refer to a specific length ofthe product. Thus, peptides, dipeptides, tripeptides, oligopeptides,“protein,” “amino acid chain,” or any other term used to refer to achain or chains of two or more amino acids, are included within thedefinition of “polypeptide,” and the terms “polypeptide” is used insteadof, or interchangeably with any of these terms.

As used herein, the terms “polypeptide” and “protein” also refer toproducts of post-expression modifications of the polypeptide, includingwithout limitation glycosylation, acetylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, or modification by non-naturally occurring aminoacids or other chemical moieties. A polypeptide can be derived from anatural biological source or produced by recombinant technology, butneed not necessarily be translated from a particular nucleic acidsequence. Polypeptides of the invention can be generated in any manner,including by chemical synthesis.

A polypeptide of the invention can be of a size of about 3 or more, 5 ormore, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 ormore, 200 or more, 500 or more, 1,000 or more, or 2,000 or more aminoacids. Polypeptides can have a defined three-dimensional structure,although they do not necessarily have such structure. Polypeptides witha defined three-dimensional structure can be referred to herein asfolded, and polypeptides which do not possess a definedthree-dimensional structure, but rather can adopt a large number ofdifferent conformations, and may be referred to herein as unfolded. Asused herein, the term glycoprotein refers to a polypeptide coupled to atleast one carbohydrate moiety that is attached to the protein via anoxygen-containing or a nitrogen-containing side chain of an amino acidresidue, e.g., a serine residue or an asparagine residue.

As used herein, an “isolated” polypeptide or a fragment, variant, orderivative thereof, refers to a polypeptide that is not in its naturalmilieu. No particular level of purification is required. For example, anisolated polypeptide can be removed from its native or naturalenvironment. Recombinantly produced polypeptides and proteins expressedin host cells can be considered isolated for the purposes of theinvention, as are native or recombinant polypeptides which have beenseparated, fractionated, or partially or substantially purified by anysuitable technique.

Also included as polypeptides of the invention are fragments,derivatives, analogs, or variants of the foregoing polypeptides, and anycombination thereof. The terms “fragment,” “variant,” “derivative” and“analog” when referring to antibodies or TDP-43 binding polypeptides ofthe invention include any polypeptides which retain at least some of theantigen-binding properties of the corresponding reference antibody orTDP-43 binding molecule. Fragments of TDP-43 binding polypeptides suchas antigen binding antibody fragments, include proteolytic fragments, aswell as deletion fragments, in addition to additional antibody fragmentsdiscussed herein. Variants of TDP-43 binding molecule, such asantibodies (including antigen binding antibody fragments) and alsopolypeptides with altered amino acid sequences due to amino acidsubstitutions, deletions, or insertions. Variants can occur naturally orbe non-naturally occurring. Non-naturally occurring variants can beproduced using art-known mutagenesis techniques. Variant polypeptidescan comprise conservative or non-conservative amino acid substitutions,deletions or additions. Derivatives of TDP-43 specific bindingmolecules, e.g., antibodies and other TDP-43 specific binding molecules,are polypeptides which have been altered so as to exhibit altered oradditional features not found on a reference polypeptide. Examples ofderivatives of TDP-43 specific binding molecules include fusionproteins. Variant polypeptides can also be referred to herein as“polypeptide analogs”. As used herein, a “derivative” of a of TDP-43specific binding molecules or fragment thereof, refers to a subjectpolypeptide having one or more residues chemically derivatized byreaction of a functional side group. Also included as “derivatives” arethose peptides which contain one or more naturally occurring amino acidderivatives of the twenty standard amino acids. For example, accordingto some embodiments, 4-hydroxyproline can be substituted for proline;5-hydroxylysine can be substituted for lysine; 3-methylhistidine can besubstituted for histidine; homoserine can be substituted for serine; orornithine can be substituted for lysine.

As used herein, the term “polynucleotide” or “nucleic acid” encompassesa singular nucleic acid as well as plural nucleic acids, and includes anisolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA)or plasmid DNA (pDNA). A polynucleotide can comprise a conventionalphosphodiester bond or a non-conventional bond (e.g., an amide bond,such as found in peptide nucleic acids (PNA)).

The term “polynucloetide acid” can also refer to any one or more nucleicacid segments, e.g., DNA or RNA fragments, present in a polynucleotide.By “isolated” nucleic acid or polynucleotide is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. For example, a recombinant polynucleotide encoding anantibody heavy or light chain variable domain contained in a vector isconsidered isolated for the purposes of the invention. Further examplesof an isolated polynucleotide include recombinant polynucleotidesmaintained in heterologous host cells or purified (partially orsubstantially) polynucleotides in solution. Isolated RNA moleculesinclude in vivo or in vitro RNA transcripts of polynucleotides of theinvention. Isolated polynucleotides or nucleic acids according to theinvention further include such molecules produced synthetically. Inaddition, polynucleotide or a nucleic acid can be or can include aregulatory element such as a promoter, ribosome binding site, or atranscription terminator operably associated with a sequence encoding aTDP-43 specific binding polypeptide of the invention.

As used herein, a “coding region” is a portion of nucleic acid whichconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it can beconsidered to be part of a coding region, but any flanking sequences,for example promoters, ribosome binding sites, transcriptionalterminators, introns, and the like, are not part of a coding region. Twoor more coding regions of the invention can be present in a singlepolynucleotide construct, e.g., on a single vector, or in separatepolynucleotide constructs, e.g., on separate (different) vectors.Furthermore, any vector can contain a single coding region, or cancomprise two or more coding regions, e.g., a single vector canseparately encode an immunoglobulin heavy chain variable region and animmunoglobulin light chain variable region. In addition, a vectorincluding a nucleic acid of the invention can optionally encode one ormore heterologous coding regions, either fused or unfused to a nucleicacid encoding a TDP-43 binding polypeptide, including for example, anantibody or a fragment, variant, or derivative thereof. Heterologouscoding regions include without limitation specialized elements ormotifs, such as a secretory signal peptide or a heterologous functionaldomain.

In certain embodiments, the polynucleotide or nucleic acid of theinvention is DNA. A polynucleotide comprising a nucleic acid whichencodes a polypeptide optionally includes a promoter and/or othertranscription or translation control elements operably associated withone or more coding regions. An operable association is present when acoding region for a gene product, e.g., a polypeptide, is associatedwith one or more regulatory sequences in such a way as to placeexpression of the gene product under the influence or control of theregulatory sequence(s). Two DNA fragments (such as a polypeptide codingregion and a promoter associated therewith) are “operably associated” or“operably linked” if induction of promoter function results in thetranscription of mRNA encoding the desired gene product and if thelinkage between the two DNA fragments does not interfere with theability of the expression regulatory sequences to direct the expressionof the gene product or prevent with the ability of the DNA template tobe transcribed. Thus, a promoter region can be operably associated witha nucleic acid encoding a polypeptide if the promoter is capable ofeffecting transcription of that nucleic acid. The promoter can be acell-specific promoter that directs substantial transcription of the DNAonly in predetermined cells. The promoter can also be constitutive orregulatable. Other transcription control elements that are optionallyoperably linked with the nucleic acids of the invention include forexample, enhancers, operators, repressors, and transcription terminationsignals, can be operably associated with the polynucleotide to directcell-specific transcription. Examples of suitable promoters and othertranscription control regions are disclosed herein or otherwise known inthe art.

A variety of transcription control regions that can be used to controlthe expression of the polynucleotides of the invention are known in theart. These include, without limitation, transcription control regionswhich function in vertebrate cells, including but not limited to,promoter and enhancer segments from cytomegaloviruses (the immediateearly promoter, in conjunction with intron-A), simian virus 40 (theearly promoter), and retroviruses (such as Rous sarcoma virus). Othertranscription control regions include but are not limed to, thosederived from vertebrate genes such as, actin, heat shock protein, bovinegrowth hormone and rabbit β-globin, as well as other sequences capableof controlling gene expression in eukaryotic cells. Additional suitabletranscription control regions include tissue-specific promoters andenhancers as well as lymphokine-inducible promoters (e.g., promotersinducible by interferons or interleukins).

Similarly, a variety of suitable translation control elements are knownto those of ordinary skill in the art. These include, but are notlimited to, ribosome binding sites, translation initiation andtermination codons, and elements derived from picornaviruses(particularly an internal ribosome entry site, or IRES, also referred toas a CITE sequence).

In other embodiments, a polynucleotide or nucleic acid of the inventionis RNA, for example, in the form of messenger RNA (mRNA).

Polynucleotide and nucleic acid coding sequences of the invention can beassociated with additional heterologous coding sequences which encodefor example, secretory or signal peptides, which direct the secretion ofa polypeptide encoded by a polynucleotide of the invention. According tothe signal hypothesis, proteins secreted by mammalian cells have asignal peptide or secretory leader sequence which is cleaved from themature protein once export of the growing protein chain across the roughendoplasmic reticulum has been initiated. Those of ordinary skill in theart are aware that polypeptides secreted by vertebrate cells generallyhave a signal peptide fused to the N-terminus of the polypeptide, whichis cleaved from the “full length” polypeptide to produce a secreted or“mature” form of the polypeptide. In certain embodiments, the nativesignal peptide, e.g., an immunoglobulin heavy chain or light chainsignal peptide is used, or a functional derivative of that sequence thatretains the ability to direct the secretion of a polypeptide to which itis operably associated. Alternatively, a heterologous mammalian signalpeptide, or a functional derivative thereof, can be used. For example,the native signal peptide sequence can be substituted with the signalpeptide sequence of human tissue plasminogen activator (TPA), mouseβ-glucuronidase, or a signal sequence derived from a secreted protein ofa preferred host cell.

Unless stated otherwise, the terms “disorder” and “disease” are usedinterchangeably herein.

A “binding molecule” as used herein relates primarily to antibodies(including TDP-43 binding antibody fragments or derivatives), but canalso refer to other proteins and polypeptides that specificallyrecognize TDP-43 including, but not limited to, hormones, receptors,ligands, major histocompatibility complex (MHC) molecules, chaperonessuch as heat shock proteins (HSPs), and cell-cell adhesion moleculessuch as, members of the cadherin, intergrin, C-type lectin andimmunoglobulin (Ig) superfamilies. Fragments, variants, and derivativesof these polypeptides that specifically recognize TDP-43 are alsoencompassed by the invention. For the sake of clarity only and withoutrestricting the scope of the invention most of the following embodimentsare discussed with respect to antibodies (including antibody fragmentsand derivatives) which represent one embodiment of the molecules andcompositions of the invention that specifically recognize TDP-43.

The terms “antibody” and “immunoglobulin” are used interchangeablyherein. An antibody or immunoglobulin is an TDP-43-binding moleculewhich comprises at least the variable domain of a heavy chain, andnormally comprises at least the variable domains of a heavy chain and alight chain. Basic immunoglobulin structures in vertebrate systems arewell understood; see, e.g., Harlow et al., Antibodies: A LaboratoryManual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988). As will bediscussed in more detail below, the term “immunoglobulin” comprisesvarious broad classes of polypeptides that can be distinguishedbiochemically. As generally understood in the art, heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, (γ, μ, α, δ, ε) withsome subclasses among them (e.g., γ1-γ4). It is the nature of this chainthat determines the “class” of the antibody as IgG, IgM, IgA, IgG, orIgE, respectively. The immunoglobulin subclasses (isotypes) e.g., IgG1,IgG2, IgG3, IgG4, IgA1, etc. are well characterized and are known toconfer functional specialization that can be incorporated or modified inthe antibodies of the invention to modify functional or other propertiesof these antibodies. Modified versions of antibody classes and isotypesof the invention are readily discernible to person of ordinary skill inthe art in view of the disclosure and are within the scope of theinvention. Additionally while all immunoglobulin classes are encompassedby the scope of the invention, for brevity and exemplary purposes, thefollowing discussion is generally directed to the IgG class ofimmunoglobulin molecules. With regard to IgG, a standard immunoglobulinmolecule comprises two identical light chain polypeptides of molecularweight approximately 23,000 Daltons, and two identical heavy chainpolypeptides of molecular weight 53,000-70,000 Daltons. The four chainsare typically joined by disulfide bonds in a “Y” configuration whereinthe light chains bracket the heavy chains starting at the mouth of the“Y” and continuing through the variable region.

Light chain polypeptides are classified as either kappa or lambda (κ,λ). Each heavy chain polypeptide class can be bound with either a kappaor lambda light chain. In general, the light and heavy chainpolypeptides are covalently bonded to each other, and the “tail”portions of the two heavy chain polypeptides are bonded to each other bycovalent disulfide linkages or non-covalent linkages when theimmunoglobulins are generated either by hybridomas, B cells orgenetically engineered host cells. In the heavy chain polypeptide, theamino acid sequences run from the N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each heavy chainpolypeptide.

Both the light and heavy chain polypeptides contain regions ofstructural and functional homology. The terms “constant” and “variable”are used functionally. In this regard, it will be appreciated that thevariable domains of both the light (V_(L)) and heavy (V_(H)) chainregions determine antigen recognition and specificity. Conversely, theconstant domains of the light chain (CL) and the heavy chain (CH1, CH2or CH3) confer important biological properties such as secretion,transplacental mobility, Fc receptor binding, complement binding, andthe like. By convention the numbering of the constant region domainsincreases as they become more distal from the antigen-binding site oramino-terminus of the antibody. The N-terminal portion of the heavy andlight chain polypeptides is a variable region and at the C-terminalportion is a constant region; the CH3 and CL domains actually comprisethe carboxy-terminus of the heavy and light chain, respectively.

As indicated above, the variable region allows the antibody toselectively recognize and specifically bind epitopes on antigens. Thatis, the V_(L) domain and V_(H) domain, or subset of the complementaritydetermining regions (CDRs), of an antibody combine to form the variableregion that defines a three dimensional antigen-binding site. Thisquaternary antibody structure forms the antigen-binding site present atthe end of each arm of the Y structure of the antibody. Theantigen-binding site of a complete antibody is defined by three CDRs oneach of the V_(H) and V_(L) chains. Any antibody (including antibodyfragments, derivatives, and variants) which contains sufficientimmunoglobulin related structure so as to allow it to specifically bindto TDP-43 is referred to herein interchangeably as a “binding fragment”or “immunospecific fragment,” and can generally be referred to asantibody that specifically recognizes TDP-43.

In naturally occurring antibodies, an antibody comprises sixhypervariable regions that are often referred to as “complementaritydetermining regions” or “CDRs” present in each antigen-binding domain.CDRs are short, non-contiguous sequences of amino acids that form theantigen-binding domain as the antibody assumes its three dimensionalconfiguration in an aqueous environment. The “CDRs” are flanked by fourrelatively conserved “framework” regions or “FRs” which show lessinter-molecular sequence variability than the CDRs. The frameworkregions largely adopt a n-sheet conformation and the CDRs form loopswhich connect, and in some cases form part of, the n-sheet structure.Thus, the framework regions act to form a scaffold that provides forpositioning the CDRs in correct orientation by inter-chain, non-covalentinteractions. The antigen-binding domain formed by the collectivelypositioned heavy and light chain CDRs defines a surface complementary tothe epitope on the immunoreactive antigen. This complementary surfacepromotes the non-covalent binding of the antibody to its cognateepitope. The amino acids comprising the CDRs and the framework regions,respectively, can be readily identified and defined for any given heavyor light chain variable region using methods known in the art; see,“Sequences of Proteins of Immunological Interest,” Kabat, E., et al.,U.S. Department of Health and Human Services, (1983); and Chothia andLesk, J. Mol. Biol., 196 (1987), 901-917, which are herein incorporatedby reference in their entireties.

In the case where there are two or more definitions of a term which isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarity determining region” (“CDR”) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. This particular region hasbeen described by Kabat et al., U.S. Dept. of Health and Human Services,“Sequences of Proteins of Immunological Interest” (1983) and by Chothiaand Lesk, J. Mol. Biol., 196 (1987), 901-917, which are hereinincorporated by reference in their entireties, where the definitionsinclude overlapping or subsets of amino acid residues when comparedagainst each other. Nevertheless, application of either definition torefer to a CDR of an antibody or variants thereof is intended to bewithin the scope of the term CDR as used herein. The appropriate aminoacid residues which encompass the CDRs as defined in each of the abovecited references are set forth in FIG. 1. The exact residue numberswhich encompass a particular CDR will vary depending on the sequence andsize of the CDR. A person of ordinary skill in the art provided with thevariable region sequence of an antibody can routinely determine whichresidues comprise a particular hypervariable region or CDR of a humanIgG antibody.

TABLE 1 CDR Definitions¹ Kabat Chothia VH CDR1 31-35 26-32 VH CDR2 50-6552-58 VH CDR3  95-102  95-102 VL CDR1 24-34 26-32 VL CDR2 50-56 50-52 VLCDR3 89-97 91-96 ¹Numbering of all CDR definitions in Table 2 isaccording to the numbering conventions set forth by Kabat et al., (seebelow).

Kabat et al., also defined a numbering system for variable domainsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable domain sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless otherwise specified, references to the numbering of specificamino acid residue positions in an antibody or antigen-binding fragment,variant, or derivative thereof of the invention are according to theKabat numbering system, which, however, is theoretical and may notequally apply to every antibody of the invention. For example, dependingon the position of the first CDR (i.e., CDR1), the following CDRs mightbe shifted in either direction.

In some embodiments, an antibody of the invention is a monoclonalantibody. In additional embodiments, an antibody of the invention is nota polyclonal antibody. According to some embodiments, an antibody of theinvention is a bivalent, or multispecific antibody. In otherembodiments, an antibody of the invention is a polyclonal antibody. Infurther embodiments, the compositions of the invention containmonoclonal antibodies. In additional embodiments, the compositions ofthe invention do not contain a polyclonal antibody.

In additional embodiments, the antibody is human (e.g., a fully humanand/or completely human antibody), humanized, primatized, murinized or achimeric antibody. In further embodiments, an antibody of the inventionis a single chain antibody, epitope-binding fragment, e.g., Fab, Fab′and F(ab′)2, Fd, Fv single-chain Fv (scFv), single-chain antibody,disulfide-linked Fv (sdFv), a fragment comprising either a V_(L) orV_(H) domain, a fragment produced by a Fab expression library, or ananti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodiesto antibodies containing a variable domain sequence provided in FIG. 1,and other antibodies disclosed herein). ScFv molecules are known in theart and are described, e.g., in U.S. Pat. No. 5,892,019. Antibodies ofthe invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, andIgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule.

In some embodiments, antibodies of the invention are IgG1. In otherembodiments, antibodies of the invention are IgG3. In a furtherembodiment, the antibody of the invention is not IgM or a derivativethereof that contains a pentavalent structure. More particularly, incertain applications of the invention, especially those relating totherapeutic use, IgMs are less desirable than IgG and other bivalentantibodies or corresponding binding molecules since IgMs often showunspecific cross-reactivities and very low affinity as a consequence oftheir pentavalent structure and lack of affinity maturation.

In a particular embodiment, the antibody of the invention is not apolyclonal antibody, i.e., it substantially consists of one particularantibody species rather than being a mixture obtained from a plasmaimmunoglobulin sample.

According to one embodiment, an antibody of the invention is a“completely” human” monoclonal antibody that specifically recognizeshuman TDP-43 and that is isolated from a human. Compared to other humanmonoclonal antibodies, such as those derived from single chain antibodyfragments (scFvs) identified using a phage display library or xenogeneicmice, completely human monoclonal antibodies of the invention arecharacterized by (i) being obtained using the human immune responserather than from animal surrogates, i.e. the antibody has been generatedin response to native endogenous TDP-43 in its relevant conformation inthe human body, (ii) having protected the individual or is at leastsignificant for the presence of TDP-43, and (iii) having a reduced risksof self-reactivity against self-antigens due to the fact that theantibody is of human origin.

Thus, while the terms “completely human antibody,” or “human monoclonalautoantibody “encompasses the terms “human antibody,” “human monoclonalantibody,” and the like, these terms are used herein to denote a TDP-43binding molecule which is of human origin, i.e. which has been derivedfrom a human antibody producing cell such as a B cell or hybridomathereof or a cell containing nucleic acids such as cDNA that is the cDNAof which has been directly cloned from or derived from a human antibodyproducing cell such as, a human memory B cell. An antibody is considered“completely human” for the purposes of this disclosure when the antibodycontains one or more amino acid substitutions or other alterations of acompletely human antibody, e.g., to improve binding characteristics.Optionally, the framework regions of the completely human antibody orother antibodies of the invention are or have been modified to conformwith a human germ line variable region sequence or to conform with aportion of a human germ line variable region sequence, such as asequence available for example, in Vbase (vbase.mrc-cpe.cam.ac.uk)hosted by the MRC Centre for Protein Engineering (Cambridge, UK). Suchmodifications can be useful, inter alia, to reduce or eliminate germline sequence deviations resulting from cloning artifacts, such as thosethat may result from PCR primers.

Antibodies derived from human immunoglobulin libraries or from animalstransgenic for one or more human are generally referred to herein ashuman antibodies, or “human-like antibodies.” Such immunoglobulins donot correspond to endogenous human immunoglobulins, as described infra.See, e.g., U.S. Pat. No. 5,939,598). For example, the pairing of heavyand light chains of human-like antibodies such as synthetic andsemi-synthetic antibodies typically isolated from phage display do notnecessarily reflect the original pairing as it occurred in the originalhuman B cell. Accordingly, Fab and scFv fragments obtained fromrecombinant expression libraries can be considered to be artificial andmay display immunogenicity and stability effects as a result of theirartificial composition. By contrast, completely human antibodies of theinvention are isolated, affinity-matured antibodies from selected humansubjects and the antibodies have been characterized by their tolerancein man.

As used herein, the term “murinized antibody” or “murinizedimmunoglobulin” refers to an antibody comprising one or more CDRs from ahuman antibody or other antibody of the invention; and for example ahuman framework region that contains amino acid substitutions and/ordeletions and/or insertions that are based on a mouse antibody sequence.In this case, the human or other immunoglobulin providing the CDRs iscalled the “parent” or “acceptor” and the mouse antibody providing theframework changes is called the “donor.” Constant regions need not bepresent, but if they are, they are usually substantially identical tomouse antibody constant regions, i.e. at least about 85-90%, or at leastabout 95%, about 97%, about 98%, or about 99% or more identical tocorresponding sequence of the mouse constant region. Hence, in someembodiments, a complete murinized human heavy or light chainimmunoglobulin contains a mouse constant region, one or more human CDRs,and a substantially human framework that has a number of “murinizing”amino acid substitutions. Typically, a “murinized antibody” is anantibody comprising a murinized variable light chain and/or a murinizedvariable heavy chain. For example, a murinized antibody would notencompass a typical chimeric antibody, e.g., because the entire variableregion of a chimeric antibody is non-mouse. A modified antibody that hasbeen “murinized” by the process of “murinization” binds to the sameantigen as the parent antibody that provides the CDRs and is usuallyless immunogenic in mice, as compared to the parent antibody.

As used herein, the term “heavy chain portion” includes amino acidsequences derived from an immunoglobulin heavy chain. A polypeptidecomprising a heavy chain portion comprises at least one of: a CH1domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain,a CH2 domain, a CH3 domain, or a variant or fragment thereof. Forexample, a binding polypeptide of the invention can comprise apolypeptide containing a variable region(s) or portions of a variableregion (e.g., one or more CDRs, such as the V_(H) CDR3), alone or incombination with a polypeptide chain comprising a CH1 domain, at least aportion of a hinge domain, and a CH2 domain; a polypeptide chaincomprising a CH1 domain and a CH3 domain; a polypeptide chain comprisinga CH1 domain, at least a portion of a hinge domain, and a CH3 domain, ora polypeptide chain comprising a CH1 domain, at least a portion of ahinge domain, a CH2 domain, and a CH3 domain. In another embodiment, apolypeptide of the invention comprises a variable region(s) or portionsof a variable region (e.g., one or more CDRs, such as, V_(H) CDR3) and apolypeptide chain comprising a CH3 domain. In a further embodiment, apolypeptide of the invention lacks at least a portion of a CH2 domain(e.g., all or part of a CH2 domain). As set forth herein, and as wouldbe appreciated by one of ordinary skill in the art, the above heavychain polypeptide domains (e.g., the heavy chain portions) can bemodified such that they vary in amino acid sequence from the naturallyoccurring immunoglobulin molecule. Accordingly, the inventionencompasses polypeptides comprising fragments, variants, and derivativesof the heavy chain portions of the invention.

According to some embodiments, the heavy chain portions of onepolypeptide chain of an antibody (including antigen-binding fragments,variants, or derivatives thereof) are identical to those on a secondpolypeptide chain of the antibody. In alternative embodiments, the heavychain portions of one polypeptide chain of an antibody (includingantigen-binding fragments, variants, or derivatives thereof) aredifferent from that on a second polypeptide chain of the antibody. Thus,each monomer component of an antibody of the invention can comprise adifferent target binding site, forming, for example, a bispecificantibody or diabody.

Antibody fragments of the invention, including single-chain antibodies,can comprise variable region(s) or portions of variable regions (e.g.,one or more CDRs, such as, VH CDR3 or VL CDR3) alone or in combinationwith the entirety or a portion of the following: hinge region, CH1, CH2,and CH3 domains. Also encompassed by the invention are TDP-43-bindingfragments that comprise any combination of variable region(s) with ahinge region, CH1, CH2, and CH3 domains. Antibodies (includingimmunospecific fragments thereof) of the invention can be derived fromany animal origin including birds and mammals. In one embodiment, theantibodies are human, murine, donkey, rabbit, goat, guinea pig, camel,llama, horse, or chicken antibodies. In another embodiment, the variableregion can be condricthoid in origin (e.g., from sharks).

In another embodiment, the antibodies disclosed herein are composed of asingle polypeptide chain such as scFvs and are to be expressedintracellularly (intrabodies) for potential in vivo therapeutic anddiagnostic applications.

The heavy chain portions or light chain portions of a bindingpolypeptide for use in the diagnostic and treatment methods disclosedherein can be derived from different immunoglobulin molecules. Forexample, a heavy chain portion of a polypeptide can comprise a CH1domain derived from an IgG1 molecule and a hinge region derived from anIgG3 molecule. In another example, a heavy chain portion can comprise ahinge region derived, in part, from an IgG1 molecule and, in part, froman IgG3 molecule. In another example, a heavy chain portion can comprisea chimeric hinge derived, in part, from an IgG1 molecule and, in part,from an IgG4 molecule.

As used herein, the term “light chain portion” includes amino acidsequences derived from an immunoglobulin light chain. In one embodiment,the light chain portion comprises at least one V_(L) or CL domain. Asused herein, the term “light chain portion” includes amino acidsequences derived from an immunoglobulin light chain. A polypeptidecomprising a light chain portion comprises at least a light chainvariable region(s) or portions of a variable region (e.g., one or moreCDRs, such as the V_(L) CDR3). In some embodiments, the light chainportion includes a CH1 domain. In another embodiment, the light chainportion comprises at least one of a V_(L) or CL domain. Polypeptidescomprising fragments, variants, and derivatives of these light chainportions are also encompassed by the invention.

The minimum size of a peptide or polypeptide epitope for an antibody isthought to be about four to five amino acids. Peptide or polypeptideepitopes can contain at least seven, at least nine, or between at leastabout 15 to about 30 amino acids. Since a CDR can recognize an antigenicpeptide or polypeptide in its tertiary form, the amino acids comprisingan epitope need not be contiguous, and in some cases, may not even be onthe same peptide chain. According to one embodiment, a peptide orpolypeptide epitope recognized by an antibody of the invention containsa sequence of at least 4, at least 5, at least 6, at least 7, at least8, at least 9, at least 10, at least 15, at least 20, at least 25contiguous or non-contiguous amino acids of TDP-43. In an additionalembodiment, a peptide or polypeptide epitope recognized by an antibodyof the invention contains between about 5 to about 30, about 10 to about30, or 15 to about 30 contiguous or non-contiguous amino acids ofTDP-43.

The terms “specifically binding” and “specifically recognizing” are usedinterchangeably herein and generally refer to a binding molecule (e.g.,a polypeptide such as an antibody) that binds to an epitope or antigenmore readily than it would bind to a random, unrelated epitope orantigen. As understood in the art, an antibody can specifically bind to,or specifically recognize an isolated polypeptide comprising, orconsisting of, amino acid residues corresponding to a linear portion ofa non-contiguous epitope. The term “specificity” is used herein toqualify the relative affinity by which a certain antibody binds to acertain epitope or antigen. For example, antibody “A” can be deemed tohave a higher specificity for a given epitope than antibody “B,” orantibody “A” can be said to bind to epitope “C” with a higherspecificity than it has for related epitope “D”. For example, antibody“A” can be deemed to have a higher specificity for a given epitope thanantibody “B,” or antibody “A” can be said to bind to epitope “C” with ahigher specificity than it has for related epitope “D.” Likewise, anantibody “A” can be deemed to have a higher specificity for a givenantigen than antibody “B,” or antibody “A” can be said to bind toantigen “C” with a higher specificity than it has for related antigen“D.”

Where present, the term “immunological binding characteristics,” orother binding characteristics of an antibody with an antigen, in all ofits grammatical forms, refers to the specificity, affinity,cross-reactivity, or other binding characteristics of an antibody.

By “preferentially binding”, it is meant that the binding molecule,e.g., antibody specifically binds to an epitope or antigen more readilythan it would bind to a related, similar, homologous, or analogousepitope or antigen. Thus, an antibody which “preferentially binds” to agiven epitope or antigen would more likely bind to that epitope orantigen than to a related epitope or antigen, even though such anantibody can cross-react with the related epitope or antigen.

By way of a non-limiting example, a binding molecule, e.g., an antibodycan be considered to bind a first epitope or antigen preferentially ifit binds said first epitope or antigen with a dissociation constant(K_(D)) that is less than the antibody's K_(D) for the second epitope orantigen. In another non-limiting example, an antibody can be consideredto bind a first antigen preferentially if it binds the first epitope orantigen with an affinity that is at least one order of magnitude lessthan the antibody's K_(D) for the second epitope or antigen. In anothernon-limiting example, an antibody can be considered to bind a firstepitope or antigen preferentially if it binds the first epitope orantigen with an affinity that is at least two orders of magnitude lessthan the antibody's K_(D) for the second epitope or antigen.

In another non-limiting example, a binding molecule, e.g., an antibodycan be considered to bind a first epitope or antigen preferentially ifit binds the first epitope or antigen with an off rate (k(off)) that isless than the antibody's k(off) for the second epitope or antigen. Inanother non-limiting example, an antibody can be considered to bind afirst epitope or antigen preferentially if it binds the first epitopewith an affinity that is at least one order of magnitude less than theantibody's k(off) for the second epitope or antigen. In anothernon-limiting example, an antibody can be considered to bind a firstepitope or antigen preferentially if it binds the first epitope orantigen with an affinity that is at least two orders of magnitude lessthan the antibody's k(off) for the second epitope or antigen.

According to one embodiment, a TDP-43 binding molecule (e.g., anantibody, including an antigen-binding fragment or variant of anantibody or derivative thereof) binds TDP-43 or a fragment or variantthereof, with an off rate (k(off)) of less than or equal to 5×10⁻²sec⁻¹, 10⁻² sec⁻¹, 5×10⁻³ sec⁻¹ or 10⁻³ sec⁻¹. In another embodiment, aTDP-43 binding molecule binds TDP-43 or a fragment or variant thereof,with an off rate (k(off)) less than or equal to 5×10⁻⁴ sec⁻¹, 10⁻⁴sec⁻¹, 5×10⁻⁵ sec⁻¹, or 10⁻⁵ sec⁻¹ 5×10⁻⁶ sec⁻¹, 10⁻⁶ sec⁻¹, 5×10⁻⁷sec⁻¹ or 10⁻⁷ sec⁻¹.

According to another embodiment, a TDP-43 binding molecule (e.g., anantibody, including an antigen-binding fragment or variant of anantibody or derivative thereof) binds TDP-43 or a fragment or variantthereof, with an on rate (k(on)) of greater than or equal to 10³ M⁻¹sec⁻¹, 5×10³ M⁻¹ sec⁻¹, 10⁴ M⁻¹ sec⁻¹ or 5×10⁴ M⁻¹ sec⁻¹. In anadditional embodiment, a TDP-43 binding molecule of the invention bindsTDP-43 or a fragment or variant thereof with an on rate (k(on)) greaterthan or equal to 10⁵ M⁻¹ sec⁻¹, 5×10⁵M⁻¹ sec⁻¹, 10⁶M⁻¹ sec⁻¹, or5×10⁶M⁻¹ sec⁻¹ or 10⁷M⁻¹ sec⁻¹.

The invention also encompasses a TDP-43 binding molecule that competeswith one or more of the TDP-43 binding molecules of the invention forbinding with TDP-43. For the purposes of this invention, a TDP-43binding molecule (e.g., an antibody) is said to competitively inhibitbinding of a reference TDP-43 binding molecule (e.g., antibody) to agiven epitope or antigen if it preferentially binds to that epitope orantigen to the extent that it blocks, to some degree, binding of thereference antibody to the epitope or antigen. Competitive inhibition canbe determined by any method known in the art, for example, competitionELISA assays. According to one embodiment, an a TDP-43 binding molecule(e.g., an antibody) competitively inhibits binding of a reference TDP-43binding molecule (e.g., an antibody) to a given epitope or antigen by atleast 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

As used herein, the term “affinity” refers to a measure of the strengthof the binding of an individual epitope or antigen with a TDP-43 bindingmolecule (e.g., an antibody, including fragments, variants, andderivatives thereof. See, e.g., Harlow et al., Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988) at pages27-28. As used herein, the term “avidity” refers to the overallstability of the complex between a population of immunoglobulins and anantigen, that is, the functional combining strength of an immunoglobulinmixture with the antigen; see, e.g., Harlow at pages 29-34. Avidity isrelated to both the affinity of individual immunoglobulin molecules inthe population with specific epitopes or antigens, and also thevalencies of the immunoglobulins and the antigen. For example, theinteraction between a bivalent monoclonal antibody and an antigen with ahighly repeating epitope structure, such as a polymer, would be one ofhigh avidity. The affinity or avidity of an antibody for an antigen canbe determined experimentally using any suitable method; see, forexample, Berzofsky et al., “Antibody-Antigen Interactions” InFundamental Immunology, Paul, W. E., Ed., Raven Press New York, N Y(1984), Kuby, Janis Immunology, W. H. Freeman and Company New York, N Y(1992), and methods described herein. General techniques for measuringthe affinity of an antibody for an antigen include ELISA, RIA, andsurface plasmon resonance. The measured affinity of a particularantibody-antigen interaction can vary if measured under differentconditions, e.g., salt concentration, pH. Thus, measurements of affinityand other antigen-binding parameters, e.g., K_(D), IC₅₀, are preferablymade with standardized solutions of antibody and antigen, and astandardized buffer.

TDP-43 binding molecules (e.g., antibodies including antigen-bindingfragments of antibodies and variants or derivatives thereof) of theinvention are also described or specified in terms of theircross-reactivity. As used herein, the term “cross-reactivity” refers tothe ability of an TDP-43 binding molecule (e.g., an antibody) specificfor one antigen, to react with a second distinct antigen; a measureoften reflective of the degree of relatedness between two differentantigenic substances.

For example, certain antibodies have some degree of cross-reactivity, inthat they bind related, but non-identical epitopes or antigens, e.g.,epitopes with at least 95%, at least 90%, at least 85%, at least 80%, atleast 75%, at least 70%, at least 65%, at least 60%, at least 55%, andat least 50% identity (as calculated using methods described herein orotherwise known in the art) to a reference epitope or antigen. Anantibody can be deemed “highly specific” for a certain epitope, if itdoes not bind any other analog, ortholog, or homolog of that epitope orantigen. According to one embodiment TDP-43 binding molecules (e.g.,antibodies including antigen-binding fragments of antibodies, andvariants or derivatives thereof) do not bind epitopes with less than95%, less than 90%, less than 85%, less than 80%, less than 75%, lessthan 70%, less than 65%, less than 60%, less than 55%, and less than 50%identity (as calculated using methods described herein or otherwiseknown in the art) to a reference epitope or antigen under physiologicalconditions.

TDP-43 binding molecules such as antibodies (including antigen-bindingfragments of an antibody and variants or derivatives thereof) of theinvention can also be described in terms of their binding affinity toTDP-43. According to one embodiment TDP-43 binding molecules (e.g.,antibodies including antigen-binding fragments, variants or derivativesthereof) binding affinities include those with a dissociation constantor Kd of less than 5×10⁻² M, 10⁻²M, 5×10⁻³M, 10⁻³M, 5×10⁻⁴M, 10⁻⁴M,5×10⁻⁵M, 10⁻⁵M, 5×10⁻⁶M, 10⁻⁶M, 5×10⁻⁷M, 10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M,10⁻⁹M, 5×10⁻¹° M, 10⁻¹⁰ M, 5×10⁻¹¹M, 10⁻¹¹M, 5×10⁻¹²M, 10⁻¹²M, 5×10⁻¹³M,10⁻¹³M, 5×10⁻¹⁴M, 10⁻¹⁴ M, 5×10⁻¹⁵M, or 10⁻¹⁵M.

The subunit structures and three dimensional configuration of theconstant regions of the various immunoglobulin classes are well known.As used herein, the term “V_(H) domain” includes the amino terminalvariable domain of an immunoglobulin heavy chain and the term “CH1domain” includes the first (most amino terminal) constant region domainof an immunoglobulin heavy chain. The CH1 domain is adjacent to theV_(H) domain and is amino terminal to the hinge region of animmunoglobulin heavy chain molecule.

As used herein the term “CH2 domain” includes the portion of a heavychain molecule that extends, e.g., from about residue 244 to residue 360of an antibody using conventional numbering schemes (residues 244 to360, Kabat numbering system; and residues 231-340, EU numbering system;see Kabat E A et al., op. cit). The CH2 domain is unique in that it isnot closely paired with another domain. Rather, two N-linked branchedcarbohydrate chains are interposed between the two CH2 domains of anintact native IgG molecule. It is also well documented that the CH3domain extends from the CH2 domain to the C-terminal of the IgG moleculeand comprises approximately 108 residues.

As used herein, the term “hinge region” includes the portion of a heavychain molecule that joins the CH1 domain to the CH2 domain. This hingeregion comprises approximately 25 residues and is flexible, thusallowing the two N-terminal antigen-binding regions to moveindependently. Hinge regions can be subdivided into three distinctdomains: upper, middle, and lower hinge domains; see Roux et al., J.Immunol. 161 (1998), 4083.

As used herein the term “disulfide bond” includes the covalent bondformed between two sulfur atoms. The amino acid cysteine comprises athiol group that can form a disulfide bond or bridge with a second thiolgroup. In most naturally occurring IgG molecules, the CH1 and CL regionsare linked by a disulfide bond and the two heavy chains are linked bytwo disulfide bonds at positions corresponding to 239 and 242 using theKabat numbering system (position 226 or 229, EU numbering system).

As used herein, the terms “linked,” “fused” or “fusion” are usedinterchangeably. These terms refer to the joining together of two moreelements or components, by whatever means including chemical conjugationor recombinant means. An “in-frame fusion” refers to the joining of twoor more polynucleotide open reading frames (ORFs) to form a continuouslonger ORF, in a manner that maintains the correct translational readingframe of the original ORFs. Thus, a recombinant fusion protein is asingle protein containing two or more segments that correspond topolypeptides encoded by the original ORFs (which segments are notnormally so joined in nature). Although the reading frame is thus madecontinuous throughout the fused segments, the segments can be physicallyor spatially separated by, for example, in-frame linker sequence. Forexample, polynucleotides encoding the CDRs of an immunoglobulin variableregion can be fused, in-frame, but be separated by a polynucleotideencoding at least one immunoglobulin framework region or additional CDRregions, as long as the “fused” CDRs are co-translated as part of acontinuous polypeptide.

The term “expression” as used herein refers to a process by which a geneproduces a biochemical, for example, an RNA or polypeptide. The processincludes any manifestation of the functional presence of the gene withinthe cell including, without limitation, gene knockdown as well as bothtransient expression and stable expression. It includes withoutlimitation transcription of the gene into messenger RNA (mRNA), transferRNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) orany other RNA product, and the translation of such mRNA intopolypeptide(s). If the final desired product is a biochemical,expression includes the creation of that biochemical and any precursors.Expression of a gene produces a “gene product.” As used herein, a geneproduct can be either a nucleic acid, e.g., a messenger RNA produced bytranscription of a gene, or a polypeptide which is translated from atranscript. Gene products described herein further include nucleic acidswith post transcriptional modifications, e.g., polyadenylation, orpolypeptides with post translational modifications, e.g., methylation,glycosylation, the addition of lipids, association with other proteinsubunits, proteolytic cleavage, and the like.

As used herein, the term “sample” refers to any biological materialobtained from a subject or patient. In one embodiment, a samplecomprises blood, cerebrospinal fluid (“CSF”), or urine. In anotherembodiment a sample comprises whole blood, plasma, B cells enriched fromblood samples, or cultured cells (e.g., B cells from a subject). Inanother embodiment a sample of the invention contains a biopsy or tissuesample including neural tissue. In a further embodiment, a sample of theinvention comprises whole cells or a lysate of the cells. Samples of theinvention, including blood samples and CSF samples can be collectedusing by methods known in the art.

As used herein, the terms “treat” or “treatment” refer to therapeutictreatment and prophylactic or preventative measures, wherein the objectis to prevent or slow down (lessen) an undesired physiological change ordisorder, such as the development of dementia. Beneficial or desiredclinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” canalso mean prolonging survival as compared to expected survival if notreceiving treatment. Those in need of treatment include those alreadywith the condition or disorder as well as those prone to have thecondition or disorder or those in which the manifestation of thecondition or disorder is to be prevented.

By “subject” or “individual” or “animal” or “patient” or “mammal,” ismeant any subject, particularly a mammalian subject, e.g., a humanpatient, for whom diagnosis, prognosis, prevention, or therapy isdesired.

II. Antibodies

Antibodies that selectively bind TDP-43 are encompassed by theinvention. As used herein, the term antibody or antibodies encompassescomplete antibodies, as well as TDP-43-binding antibody fragments andvariants and derivatives of these complete antibodies or antibodyfragments that bind TDP-43. In one embodiment, an antibody of theinvention demonstrates at least one, two, three, four, five or more ofthe structural characteristics (e.g., sequence), immunological bindingcharacteristics (e.g., IC₅₀, or epitope binding), and/or biologicalproperties of the antibodies disclosed in the Examples and elsewhere inthe specification.

According to some embodiments, the antibody of the invention is acompletely human antibody. The invention also encompasses fragments,variants and derivatives of a completely human antibody. As disclosed inthe Examples, the completely human antibodies disclosed herein werederived from a pool of samples from healthy subjects. Antibodies ofpotential interest were analyzed for class and light chain subclassdetermination, message from selected memory B cell cultures weretranscribed by RT-PCR, cloned and combined into expression vectors forrecombinant production; see the appended Examples. The completely humanantibodies were then recombinantly expressed in HEK293 cells andsubsequently characterized based on their binding specificities towardsfull-length TDP-43, truncated TDP-43 and a modified form of TDP-43(FIGS. 2, 4-7, 10, and 11). This characterization confirmed that for thefirst time, human antibodies have been cloned that are highly specificfor TDP-43 and recognize different epitopes within the TDP-43 protein.

Thus, according to one embodiment, the invention generally relates to anantibody that specifically recognizes TDP-43. In a further embodiment,the invention is directed to a human antibody that specificallyrecognizes TDP-43. In yet a further embodiment, the invention isdirected to a total human antibody that specifically recognizes TDP-43.In additional embodiments, the invention encompasses a TDP-43 bindingfragment, variant or derivative of a TDP-43 binding intact human(including completely human) antibody of the invention. In anotherembodiment, the antibodies of the invention specifically recognize fulllength, truncated, or pathologic human TDP-43 in a Western Blot. In afurther embodiment, the antibodies of the invention selectively bindfull length or pathologic TDP-43 in a Western Blot. In anotherembodiment, the antibodies of the invention specifically recognize fulllength, truncated, or pathologic human TDP-43 in an ELISA. In a furtherembodiment, the antibodies of the invention selectively bind full lengthor pathologic TDP-43 in an ELISA. In another embodiment, the antibodiesof the invention specifically recognize any combination of full length,truncated, or pathologic human TDP-43 in an immunohistochemistry. In afurther embodiment, the antibodies of the invention selectively bindfull length or pathologic TDP-43 in an immunohistochemistry. In anotherembodiment, the antibodies of the invention specifically recognize anycombination of full length, truncated, or pathologic human TDP-43 in animmunohistochemistry of the hippocampus. In a further embodiment, theantibodies of the invention selectively bind full length or pathologicTDP-43 in an immunohistochemistry of the hippocampus. In a furtherembodiment, the antibodies of the invention selectively bind to one ormore of nuclear TDP-43, cytoplasmic TDP-43, axonal TDP-43, or neuriticTDP-43 in an immunohistochemistry of human FTLD-U hippocampus. Inanother embodiment, the antibodies of the invention selectively bind toone or more of cytoplasmic TDP-43 and neuritic TDP-43 in hippocampalgranule cells in an immunohistochemistry of human FTLD-U hippocampus.According to one embodiment, the antibodies of the inventionspecifically recognize pathologic human-TDP-43.

In another embodiment, the invention encompasses an antibody (includingan antigen-binding fragment, variant or derivatives thereof), thatspecifically binds to the same epitope of TDP-43 as a reference antibodyhaving the heavy and light chain variable domain of an antibody selectedfrom the group consisting of: NI-205.3F10, NI-205.51C1, NI-205.21G2,NI-205.8A2, NI-205.15F12, NI-205.113C4, NI-205.25F3, NI-205.87E7,NI-205.21G1, NI-205.68G5, NI-205.20A1, NI205.41D1, NI205.29E11,N1205.9E12, NI205.98H6, NI205.10D3, NI205.44B2, NI205.38H2, NI205.36D5,NI205.58E11, NI205.14H5, NI205.31D2, NI205.8F8, NI205.31C11, NI205.8C10,NI205.10H7, NI205.1A9, NI205.14W3, and NI205.19G5. In a furtherembodiment, the antibody (including an antigen-binding fragment, variantor derivatives thereof), specifically binds to the same epitope ofTDP-43 as a reference antibody selected from the group consisting of:NI-205.3F10, NI-205.51C1, NI-205.21G2, NI-205.8A2, NI-205.15F12,NI-205.113C4, NI-205.25F3, NI-205.87E7, NI-205.21G1, NI-205.68G5,NI-205.20A1, NI205.41D1, NI205.29E11, NI205.9E12, NI205.98H6,NI205.10D3, NI205.44B2, NI205.38H2, NI205.36D5, NI205.58E11, NI205.14H5,NI205.31D2, NI205.8F8, NI205.31C11, NI205.8C10, NI205.10H7, NI205.1A9,NI205.14W3, and NI205.19G5.

In another embodiment, the invention encompasses an antibody (includingan antigen-binding fragment, variant or derivatives thereof), thatspecifically binds to a TDP-43 polypeptide sequence selected from:QYGDVMDVFIP (SEQ ID NO: 123); AAIGWGSASNA (SEQ ID NO: 124); DMTEDELREFF(SEQ ID NO: 125), EDENDEP (SEQ ID NO: 126), VQVKKDL (SEQ ID NO: 127),KEYFSTF (SEQ ID NO: 128), IIKGISV (SEQ ID NO:315), NQSGPSG (SEQ IDNO:316), FNGGFGS (SEQ ID NO:317), FGNSRGGGAGL (SEQ ID NO:318),SNAGSGSGFNG (SEQ ID NO:319), QLERSGRFGGN (SEQ ID NO:320), EIPSEDD (SEQID NO:321), FNGGFGS SMDS (SEQ ID NO:322) and SINPAMMAAAQAALQSSWGMMGMLASQ(SEQ ID NO:323). In another embodiment, the invention encompasses anantibody (including an antigen-binding fragment, variant or derivativesthereof), that specifically binds to TDP-43 polypeptides FGNSRGGGAGL(SEQ ID NO:318) and SNAGSGSGFNG (SEQ ID NO:319). In another embodiment,the invention encompasses an antibody (including an antigen-bindingfragment, variant or derivatives thereof), that specifically binds toTDP-43 polypeptide SINPAMMAAAQAALQSSWGMMGMLASQ (SEQ ID NO:323), but doesnot specifically bind to SINPGGGAAAQAALQSSWGMMGMLASQ (SEQ ID NO:314).

In a further embodiment, the invention encompasses an antibody(including an antigen-binding fragment, variant or derivatives thereof),that competitively inhibits the binding to TDP-43 by a referenceantibody having the heavy and light chain variable domain of an antibodyselected from the group consisting of: NI-205.3F10, NI-205.51C1,NI-205.21G2, NI-205.8A2, NI-205.15F12, NI-205.113C4, NI-205.25F3,NI-205.87E7, NI-205.21G1, NI-205.68G5, NI-205.20A1, NI205.41D1,NI205.29E11, NI205.9E12, NI205.98H6, NI205.10D3, NI205.44B2, NI205.38H2,NI205.36D5, NI205.58E11, NI205.14H5, NI205.31D2, NI205.8F8, NI205.31C11,NI205.8C10, NI205.10H7, NI205.1A9, NI205.14W3, and NI205.19G5.

In a further embodiment, the antibody (including an antigen-bindingfragment, variant or derivatives thereof), competitively inhibits thebinding to TDP-43 by a reference antibody selected from the groupconsisting of: NI-205.3F10, NI-205.51C1, NI-205.21G2, NI-205.8A2,NI-205.15F12, NI-205.113C4, NI-205.25F3, NI-205.87E7, NI-205.21G1,NI-205.68G5, NI-205.20A1, NI205.41D1, NI205.29E11, NI205.9E12,NI205.98H6, NI205.10D3, NI205.44B2, NI205.38H2, NI205.36D5, NI205.58E11,NI205.14H5, NI205.31D2, NI205.8F8, NI205.31C11, NI205.8C10, NI205.10H7,NI205.1A9, NI205.14W3, and NI205.19G5.

As illustrated in the Examples, the invention encompasses antibodiesthat bind to different portions and epitopes of TDP-43. According tosome embodiments, an antibody of the invention binds a linear epitope ofTDP-43. According to other embodiments, an antibody of the inventionbinds to a conformational epitope of TDP-43. In an additional embodimentan antibody of the invention selectively binds a TDP-43 domain selectedfrom the group consisting of: TDP-43 domain I (amino acid residues 2-106of SEQ ID NO:94), TDP-43 domain II (amino acid residues 99-204 of SEQ IDNO:94), TDP-43 domain III (amino acid residues 183-273 of SEQ ID NO:94),and TDP-43 domain IV (amino acid residues 258-414 of SEQ ID NO:94). Inanother embodiment, the anti-TDP-43 antibody does not recognize atruncated form of TDP-43. In an additional embodiment, the inventionprovides an anti-TDP-43 antibody which recognizes an N-terminal₁₋₂₅₉fragment of TDP-43. In a further embodiment, the anti-TDP-43 antibodyselectively binds pathologic TDP-43. In a further embodiment, ananti-TDP-43 antibody specifically binds TDP-43 domain IV (amino acidresidues 258-414 of SEQ ID NO:94), but does not specifically bindsTDP-43 domain IV comprising the A321G, M322G, and M323G substitutions.

The invention encompasses human anti-TDP-43 antibodies having differentTPD-43 specificities, which are thus particularly useful for diagnosticand therapeutic purposes. The invention is also drawn to an antibodycomprising an antigen-binding domain having an amino acid sequenceselected from that present in a reference antibody selected from thegroup consisting of: NI-205.3F10, NI-205.51C1, NI-205.21G2, NI-205.8A2,NI-205.15F12, NI-205.113C4, NI-205.25F3, NI-205.87E7, NI-205.21G1,NI-205.68G5, NI-205.20A1, NI205.41D1, NI205.29E11, NI205.9E12,NI205.98H6, NI205.10D3, NI205.44B2, NI205.38H2, NI205.36D5, NI205.58E11,NI205.14H5, NI205.31D2, NI205.8F8, NI205.31C11, NI205.8C10, NI205.10H7,NI205.1A9, NI205.14W3, and NI205.19G5.

The examples and Figures disclose TDP-43 binding molecules that arecharacterized by containing in their binding domain at least onecomplementarity determining region (CDR) of the V_(H) and/or V_(L)variable region comprising any one of the amino acid sequences depictedin FIGS. 1A-1K and 3A-3R and listed in Table 2. The correspondingnucleotide sequences encoding these variable regions are set forth inTable 3. An exemplary set of CDRs of the above amino acid sequences ofthe V_(H) and/or V_(L) region is depicted in FIGS. 1A-1K and 3A-3R.However, as would be understood by a person of ordinary skill in theart, additional or alternative CDRs can be used, which specifically bindTDP-43, but which differ in their amino acid sequence from those setforth in FIGS. 1A-1K and 3A-3R by one, two, three or even more aminoacids in case of CDR2 and CDR3.

TABLE 2 SEQ ID NOs of the V_(H) region, V_(H) CDR1, V_(H) CDR2, V_(H)CDR2, V_(L) region, V_(L) CDR2, V_(L) CDR2, and V_(L) CDR3 of TDP-43specific antibodies. Antibody V_(H)/V_(L) CDR1 CDR2 CDR3 NI-205.3F10V_(H) SEQ ID NO: 1 SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO: 5 V_(L) SEQ IDNO: 6 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 9 NI-205.51C1 V_(H) SEQ IDNO: 10 SEQ ID NO: 11 SEQ ID NO: 12 SEQ ID NO: 13 V_(L) SEQ ID NO: 14 SEQID NO: 15 SEQ ID NO: 16 SEQ ID NO: 17 NI-205.21G2 V_(H) SEQ ID NO: 18SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO: 21 V_(L) SEQ ID NO: 22 SEQ ID NO:23 SEQ ID NO: 24 SEQ ID NO: 25 NI-205.8A2 V_(H) SEQ ID NO: 26 SEQ ID NO:28 SEQ ID NO: 29 SEQ ID NO: 30 V_(L) SEQ ID NO: 31 SEQ ID NO: 32 SEQ IDNO: 33 SEQ ID NO: 34 NI-205.15F12 V_(H) SEQ ID NO:: 35 SEQ ID NO: 37 SEQID NO: 38 SEQ ID NO: 39 V_(L) SEQ ID NO: 40 SEQ ID NO: 42 SEQ ID NO: 43SEQ ID NO: 44 NI-205.113C4 V_(H) SEQ ID NO: 45 SEQ ID NO: 46 SEQ ID NO:47 SEQ ID NO: 48 V_(L) SEQ ID NO: 49 SEQ ID NO: 50 SEQ ID NO: 51 SEQ IDNO: 52 NI-205.25F3 V_(H) SEQ ID NO: 53 SEQ ID NO: 54 SEQ ID NO: 55 SEQID NO: 56 V_(L) SEQ ID NO: 57 SEQ ID NO: 58 SEQ ID NO: 59 SEQ ID NO: 60NI-205.87E7 V_(H) SEQ ID NO: 61 SEQ ID NO: 62 SEQ ID NO: 63 SEQ ID NO:64 V_(L) SEQ ID NO: 65 SEQ ID NO: 66 SEQ ID NO: 67 SEQ ID NO: 68NI-205.21G1 V_(H) SEQ ID NO: 69 SEQ ID NO: 70 SEQ ID NO: 71 SEQ ID NO:72 V_(L) SEQ ID NO: 73 SEQ ID NO: 74 SEQ ID NO: 75 SEQ ID NO: 76NI-205.68G5 V_(H) SEQ ID NO: 77 SEQ ID NO: 79 SEQ ID NO: 80 SEQ ID NO:81 V_(L) SEQ ID NO: 82 SEQ ID NO: 84 SEQ ID NO: 85 SEQ ID NO: 86NI-205.20A1 V_(H) SEQ ID NO: 87 SEQ ID NO: 88 SEQ ID NO: 89 SEQ ID NO:90 V_(L) SEQ ID NO: 122 SEQ ID NO: 91 SEQ ID NO: 92 SEQ ID NO: 93NI205.41D1 V_(H) SEQ ID NO: 130 SEQ ID NO: 131 SEQ ID NO: 132 SEQ ID NO:133 V_(L) SEQ ID NO: 134 SEQ ID NO: 135 SEQ ID NO: 136 SEQ ID NO: 137NI205.29E11 V_(H) SEQ ID NO: 138 SEQ ID NO: 139 SEQ ID NO: 140 SEQ IDNO: 141 V_(L) SEQ ID NO: 142 SEQ ID NO: 143 SEQ ID NO: 144 SEQ ID NO:145 NI205.9E12 V_(H) SEQ ID NO: 146 SEQ ID NO: 147 SEQ ID NO: 148 SEQ IDNO: 149 V_(L) SEQ ID NO: 150 SEQ ID NO: 326 SEQ ID NO: 327 SEQ ID NO:328 V_(L) SEQ ID NO: 151 SEQ ID NO: 152 SEQ ID NO: 153 SEQ ID NO: 154NI205.98H6 V_(H) SEQ ID NO: 155 SEQ ID NO: 156 SEQ ID NO: 157 SEQ ID NO:158 V_(L) SEQ ID NO: 159 SEQ ID NO: 160 SEQ ID NO: 161 SEQ ID NO: 162NI205.10D3 V_(H) SEQ ID NO: 163 SEQ ID NO: 164 SEQ ID NO: 165 SEQ ID NO:166 V_(L) SEQ ID NO: 167 SEQ ID NO: 168 SEQ ID NO: 169 SEQ ID NO: 170NI205.44B22 V_(H) SEQ ID NO: 171 SEQ ID NO: 172 SEQ ID NO: 173 SEQ IDNO: 174 V_(L) SEQ ID NO: 175 SEQ ID NO: 176 SEQ ID NO: 177 SEQ ID NO:178 NI205.38H2 V_(H) SEQ ID NO: 179 SEQ ID NO: 180 SEQ ID NO: 181 SEQ IDNO: 182 V_(L) SEQ ID NO: 183 SEQ ID NO: 184 SEQ ID NO: 185 SEQ ID NO:186 NI205.36D5 V_(H) SEQ ID NO: 187 SEQ ID NO: 188 SEQ ID NO: 189 SEQ IDNO: 190 V_(L) SEQ ID NO: 191 SEQ ID NO: 192 SEQ ID NO: 193 SEQ ID NO:194 NI205.58E11 V_(H) SEQ ID NO: 195 SEQ ID NO: 196 SEQ ID NO: 197 SEQID NO: 198 V_(L) SEQ ID NO: 199 SEQ ID NO: 200 SEQ ID NO: 201 SEQ ID NO:202 NI205.14H5 V_(H) SEQ ID NO: 203 SEQ ID NO: 204 SEQ ID NO: 205 SEQ IDNO: 206 V_(L) SEQ ID NO: 207 SEQ ID NO: 208 SEQ ID NO: 209 SEQ ID NO:210 NI205.31D2 V_(H) SEQ ID NO: 211 SEQ ID NO: 212 SEQ ID NO: 213 SEQ IDNO: 214 V_(L) SEQ ID NO: 215 SEQ ID NO: 216 SEQ ID NO: 217 SEQ ID NO:218 NI205.8F8 V_(H) SEQ ID NO: 219 SEQ ID NO: 220 SEQ ID NO: 221 SEQ IDNO: 222 V_(L) SEQ ID NO: 223 SEQ ID NO: 224 SEQ ID NO: 225 SEQ ID NO:226 NI205.31C11 V_(H) SEQ ID NO: 227 SEQ ID NO: 228 SEQ ID NO: 229 SEQID NO: 230 V_(L) SEQ ID NO: 231 SEQ ID NO: 232 SEQ ID NO: 233 SEQ ID NO:234 NI205.8C10 V_(H) SEQ ID NO: 235 SEQ ID NO: 236 SEQ ID NO: 237 SEQ IDNO: 238 V_(L) SEQ ID NO: 239 SEQ ID NO: 240 SEQ ID NO: 241 SEQ ID NO:242 NI205.10H7 V_(H) SEQ ID NO: 243 SEQ ID NO: 244 SEQ ID NO: 245 SEQ IDNO: 246 V_(L) SEQ ID NO: 247 SEQ ID NO: 248 SEQ ID NO: 249 SEQ ID NO:250 NI205.1A9 V_(H) SEQ ID NO: 251 SEQ ID NO: 252 SEQ ID NO: 253 SEQ IDNO: 254 V_(L) SEQ ID NO: 255 SEQ ID NO: 256 SEQ ID NO: 257 SEQ ID NO:258 NI205.14W3 V_(H) SEQ ID NO: 259 SEQ ID NO: 260 SEQ ID NO: 261 SEQ IDNO: 262 V_(L) SEQ ID NO: 263 SEQ ID NO: 264 SEQ ID NO: 265 SEQ ID NO:266 NI205.19G5 V_(H) SEQ ID NO: 267 SEQ ID NO: 268 SEQ ID NO: 269 SEQ IDNO: 270 V_(L) SEQ ID NO: 271 SEQ ID NO: 272 SEQ ID NO: 273 SEQ ID NO:274

In one embodiment, an antibody of the invention comprises at least oneCDR comprising, or consisting of an amino acid sequence selected fromthe group consisting of SEQ ID NO: 3-5, 7-9, 11-13, 15-17, 19-21, 23-25,28-30, 32-34, 37-39, 42-44, 46-48, 50-52, 54-56, 58-60, 62-64, 66-68,70-72, 74-76, 79-81, 84-86, 88-93, 131-133, 135-137, 139-141, 143-145,147-149, 152-154, 156-158, 160-162, 164-166, 168-170, 172-174, 176-178,180-182, 184-186, 188-190, 192-194, 196-198, 200-202, 204-206, 208-210,212-214, 216-218, 220-222, 224-226, 228-230, 232-234, 236-238, 240-242,244-246, 248-250, 252-254, 256-258, 260-262, 264-266, 268-270, 272-274and 326-328.

In one embodiment, an antibody of the invention comprises one, two,three, four, five or six CDRs comprising, or consisting of an amino acidsequence selected from the group consisting of SEQ ID NO: 3-5, 7-9,11-13, 15-17, 19-21, 23-25, 28-30, 32-34, 37-39, 42-44, 46-48, 50-52,54-56, 58-60, 62-64, 66-68, 70-72, 74-76, 79-81, 84-86, 88-93, 131-133,135-137, 139-141, 143-145, 147-149, 152-154, 156-158, 160-162, 164-166,168-170, 172-174, 176-178, 180-182, 184-186, 188-190, 192-194, 196-198,200-202, 204-206, 208-210, 212-214, 216-218, 220-222, 224-226, 228-230,232-234, 236-238, 240-242, 244-246, 248-250, 252-254, 256-258, 260-262,264-266, 268-270, 272-274 and 326-328.

In one embodiment, an antibody of the invention comprises one, two,three, four, five or six CDRs comprising, or consisting of an amino acidsequence selected from the group consisting of SEQ ID NO: 3-5 and 7-9,11-13 and 15-17, 19-21 and 23-25, 28-30 and 32-34, 37-39 and 42-44,46-48 and 50-52, 54-56 and 58-60, 62-64 and 66-68, 70-72 and 74-76,79-81 and 84-86, 88-93, 131-133 and 135-137, 139-141 and 143-145,147-149 and 152-154, 156-158 and 160-162, 164-166 and 168-170, 172-174and 176-178, 180-182 and 184-186, 188-190 and 192-194, 196-198 and200-202, 204-206 and 208-210, 212-214 and 216-218, 220-222 and 224-226,228-230 and 232-234, 236-238 and 240-242, 244-246 and 248-250, 252-254and 256-258, 260-262 and 264-266, 268-270 and 272-274, and 147-149 and326-328.

In one embodiment, an antibody of the invention comprises one, two, orthree VH CDRs comprising, or consisting of an amino acid sequenceselected from the group consisting of SEQ ID NO: 3-5, 11-13, 19-21,28-30, 37-39, 46-48, 54-56, 62-64, 70-72, 79-81, 88-90, 131-133,139-141, 147-149, 156-158, 164-166, 172-174, 180-182, 188-190, 196-198,204-206, 212-214, 220-222, 228-230, 236-238, 244-246, 252-254, 260-262,and 268-270.

In one embodiment, an antibody of the invention comprises one, two, orthree VL CDRs comprising, or consisting of an amino acid sequenceselected from the group consisting of SEQ ID NO: 7-9, 15-17, 23-25,32-34, 42-44, 50-52, 58-60, 66-68, 74-76, 84-86, 91-93, 135-137,143-145, 152-154, 160-162, 168-170, 176-178, 184-186, 192-194, 200-202,208-210, 216-218, 224-226, 232-234, 240-242, 248-250, 256-258, 264-266,272-274 and 326-328.

According to one embodiment, an antibody of the invention comprises aheavy chain variable region comprising a VH CDR1 of SEQ ID NO: 3, 11,19, 28, 37, 46, 54, 62, 70, 79, 88, 131, 139, 147, 156, 164, 172, 180,188, 196, 204, 212, 220, 228, 236, 244, 252, 260, or 268; a VH CDR2 ofSEQ ID NO: 4, 12, 20, 29, 38, 47, 55, 63, 71, 80, 89, 132, 140, 148,157, 165, 173, 181, 189, 197, 205, 213, 221, 229, 237, 245, 253, 261, or269; or a VH CDR3 of SEQ ID NO: 5, 13, 21, 30, 39, 48, 56, 64, 72, 81,90, 133, 141, 149, 158, 166, 174, 182, 190, 198, 206, 214, 222, 230,238, 246, 254, 262, or 270. According to another embodiment, an antibodycomprises a light chain variable region comprising a VL CDR1 of SEQ IDNO: 7, 15, 23, 32, 42, 50, 58, 66, 74, 84, 91, 135, 143, 152, 160, 168,176, 184, 192, 200, 208, 216, 224, 232, 240, 248, 256, 264, 272 or 326;a VL CDR2 of SEQ ID NO: 8, 16, 24, 33, 43, 51, 59, 67, 75, 85, 92, 136,144, 153, 161, 169, 177, 185, 193, 201, 209, 217, 225, 233, 241, 249,257, 265, 273 or 327; or a VL CDR3 of SEQ ID NO: 9, 17, 25, 34, 44, 52,60, 68, 76, 86, 93, 137, 145, 154, 162, 170, 178, 186, 194, 202, 210,218, 226, 234, 242, 250, 258, 266, 274 or 328. In another embodiment,the antibody comprises a heavy chain variable region comprising a VHCDR1 of SEQ ID NO: 3, 11, 19, 28, 37, 46, 54, 62, 70, 79, 88, 131, 139,147, 156, 164, 172, 180, 188, 196, 204, 212, 220, 228, 236, 244, 252,260, or 268; a VH CDR2 of SEQ ID NO: 4, 12, 20, 29, 38, 47, 55, 63, 71,80, 89, 132, 140, 148, 157, 165, 173, 181, 189, 197, 205, 213, 221, 229,237, 245, 253, 261, or 269; or a VH CDR3 of SEQ ID NO: 5, 13, 21, 30,39, 48, 56, 64, 72, 81, 90, 133, 141, 149, 158, 166, 174, 182, 190, 198,206, 214, 222, 230, 238, 246, 254, 262, or 270, and further comprises alight chain variable region comprising a VL CDR1 of SEQ ID NO: 7, 15,23, 32, 42, 50, 58, 66, 74, 84, 91, 135, 143, 152, 160, 168, 176, 184,192, 200, 208, 216, 224, 232, 240, 248, 256, 264, 272 or 326; a VL CDR2of SEQ ID NO: 8, 16, 24, 33, 43, 51, 59, 67, 75, 85, 92, 136, 144, 153,161, 169, 177, 185, 193, 201, 209, 217, 225, 233, 241, 249, 257, 265,273 or 327; or a VL CDR3 of SEQ ID NO: 9, 17, 25, 34, 44, 52, 60, 68,76, 86, 93, 137, 145, 154, 162, 170, 178, 186, 194, 202, 210, 218, 226,234, 242, 250, 258, 266, 274 or 328.

According to one embodiment, an antibody of the invention comprises aheavy chain variable region comprising a VH CDR1 of SEQ ID NO: 3, 11,19, 28, 37, 46, 54, 62, 70, 79, 88, 131, 139, 147, 156, 164, 172, 180,188, 196, 204, 212, 220, 228, 236, 244, 252, 260, or 268; a VH CDR2 ofSEQ ID NO: 4, 12, 20, 29, 38, 47, 55, 63, 71, 80, 89, 132, 140, 148,157, 165, 173, 181, 189, 197, 205, 213, 221, 229, 237, 245, 253, 261, or269; and a VH CDR3 of SEQ ID NO: 5, 13, 21, 30, 39, 48, 56, 64, 72, 81,90, 133, 141, 149, 158, 166, 174, 182, 190, 198, 206, 214, 222, 230,238, 246, 254, 262, or 270. According to another embodiment, an antibodycomprises a light chain variable region comprising a VL CDR1 of SEQ IDNO: 7, 15, 23, 32, 42, 50, 58, 66, 74, 84, 91, 135, 143, 152, 160, 168,176, 184, 192, 200, 208, 216, 224, 232, 240, 248, 256, 264, 272 or 326;a VL CDR2 of SEQ ID NO: 8, 16, 24, 33, 43, 51, 59, 67, 75, 85, 92, 136,144, 153, 161, 169, 177, 185, 193, 201, 209, 217, 225, 233, 241, 249,257, 265, 273 or 327; and a VL CDR3 of SEQ ID NO: 9, 17, 25, 34, 44, 52,60, 68, 76, 86, 93, 137, 145, 154, 162, 170, 178, 186, 194, 202, 210,218, 226, 234, 242, 250, 258, 266, 274 or 328. In another embodiment,the antibody comprises a heavy chain variable region comprising a VHCDR1 of SEQ ID NO: 3, 11, 19, 28, 37, 46, 54, 62, 70, 79, 88, 131, 139,147, 156, 164, 172, 180, 188, 196, 204, 212, 220, 228, 236, 244, 252,260, or 268; a VH CDR2 of SEQ ID NO: 4, 12, 20, 29, 38, 47, 55, 63, 71,80, 89, 132, 140, 148, 157, 165, 173, 181, 189, 197, 205, 213, 221, 229,237, 245, 253, 261, or 269; and a VH CDR3 of SEQ ID NO: 5, 13, 21, 30,39, 48, 56, 64, 72, 81, 90, 133, 141, 149, 158, 166, 174, 182, 190, 198,206, 214, 222, 230, 238, 246, 254, 262, or 270, and further comprises alight chain variable region comprising a VL CDR1 of SEQ ID NO: 7, 15,23, 32, 42, 50, 58, 66, 74, 84, 91, 135, 143, 152, 160, 168, 176, 184,192, 200, 208, 216, 224, 232, 240, 248, 256, 264, 272 or 326; a VL CDR2of SEQ ID NO: 8, 16, 24, 33, 43, 51, 59, 67, 75, 85, 92, 136, 144, 153,161, 169, 177, 185, 193, 201, 209, 217, 225, 233, 241, 249, 257, 265,273 or 327; and a VL CDR3 of SEQ ID NO: 9, 17, 25, 34, 44, 52, 60, 68,76, 86, 93, 137, 145, 154, 162, 170, 178, 186, 194, 202, 210, 218, 226,234, 242, 250, 258, 266, 274 or 328.

In one embodiment, an antibody of the invention can comprise a heavychain variable region comprising a VH CDR1 of SEQ ID NO: 3, a VH CDR2 ofSEQ ID NO: 4, and VH CDR3 of SEQ ID NO: 5, and can further comprise alight chain variable region comprising a VL CDR1 of SEQ ID NO:7, a VLCDR2 of SEQ ID NO: 8, and a VL CDR3 of SEQ ID NO: 9.

In one embodiment, an antibody of the invention can comprise a heavychain variable region comprising a VH CDR1 of SEQ ID NO: 11, a VH CDR2of SEQ ID NO: 12, and VH CDR3 of SEQ ID NO: 13, and can further comprisea light chain variable region comprising a VL CDR1 of SEQ ID NO:15, a VLCDR2 of SEQ ID NO: 16, and a VL CDR3 of SEQ ID NO: 17.

In one embodiment, an antibody of the invention can comprise a heavychain variable region comprising a VH CDR1 of SEQ ID NO: 19, a VH CDR2of SEQ ID NO: 20, and VH CDR3 of SEQ ID NO: 21, and can further comprisea light chain variable region comprising a VL CDR1 of SEQ ID NO:23, a VLCDR2 of SEQ ID NO: 24, and a VL CDR3 of SEQ ID NO: 25.

In one embodiment, an antibody of the invention can comprise a heavychain variable region comprising a VH CDR1 of SEQ ID NO: 28, a VH CDR2of SEQ ID NO: 29, and VH CDR3 of SEQ ID NO: 30, and can further comprisea light chain variable region comprising a VL CDR1 of SEQ ID NO:32, a VLCDR2 of SEQ ID NO: 33, and a VL CDR3 of SEQ ID NO: 34.

In one embodiment, an antibody of the invention can comprise a heavychain variable region comprising a VH CDR1 of SEQ ID NO: 37, a VH CDR2of SEQ ID NO: 38, and VH CDR3 of SEQ ID NO: 39, and can further comprisea light chain variable region comprising a VL CDR1 of SEQ ID NO:42, a VLCDR2 of SEQ ID NO: 43, and a VL CDR3 of SEQ ID NO: 44.

In one embodiment, an antibody of the invention can comprise a heavychain variable region comprising a VH CDR1 of SEQ ID NO: 46, a VH CDR2of SEQ ID NO: 47, and VH CDR3 of SEQ ID NO: 48, and can further comprisea light chain variable region comprising a VL CDR1 of SEQ ID NO:50, a VLCDR2 of SEQ ID NO: 51, and a VL CDR3 of SEQ ID NO: 52.

In one embodiment, an antibody of the invention can comprise a heavychain variable region comprising a VH CDR1 of SEQ ID NO: 54, a VH CDR2of SEQ ID NO: 55, and VH CDR3 of SEQ ID NO: 56, and can further comprisea light chain variable region comprising a VL CDR1 of SEQ ID NO: 58, aVL CDR2 of SEQ ID NO: 59, and a VL CDR3 of SEQ ID NO: 60.

In one embodiment, an antibody of the invention can comprise a heavychain variable region comprising a VH CDR1 of SEQ ID NO: 62, a VH CDR2of SEQ ID NO: 63, and VH CDR3 of SEQ ID NO: 64, and can further comprisea light chain variable region comprising a VL CDR1 of SEQ ID NO:66, a VLCDR2 of SEQ ID NO: 67, and a VL CDR3 of SEQ ID NO: 68.

In one embodiment, an antibody of the invention can comprise a heavychain variable region comprising a VH CDR1 of SEQ ID NO: 70, a VH CDR2of SEQ ID NO: 71, and VH CDR3 of SEQ ID NO: 72, and can further comprisea light chain variable region comprising a VL CDR1 of SEQ ID NO:74, a VLCDR2 of SEQ ID NO: 75, and a VL CDR3 of SEQ ID NO: 76.

In one embodiment, an antibody of the invention can comprise a heavychain variable region comprising a VH CDR1 of SEQ ID NO: 79, a VH CDR2of SEQ ID NO: 80, and VH CDR3 of SEQ ID NO: 81, and can further comprisea light chain variable region comprising a VL CDR1 of SEQ ID NO:84, a VLCDR2 of SEQ ID NO: 85, and a VL CDR3 of SEQ ID NO: 86.

In one embodiment, an antibody of the invention can comprise a heavychain variable region comprising a VH CDR1 of SEQ ID NO: 88, a VH CDR2of SEQ ID NO: 89, and VH CDR3 of SEQ ID NO: 90, and can further comprisea light chain variable region comprising a VL CDR1 of SEQ ID NO:91, a VLCDR2 of SEQ ID NO: 92, and a VL CDR3 of SEQ ID NO: 93.

In one embodiment, an antibody of the invention can comprise a heavychain variable region comprising a VH CDR1 of SEQ ID NO: 131, a VH CDR2of SEQ ID NO: 132, and VH CDR3 of SEQ ID NO: 133, and can furthercomprise a light chain variable region comprising a VL CDR1 of SEQ IDNO:135, a VL CDR2 of SEQ ID NO: 136, and a VL CDR3 of SEQ ID NO: 137.

In one embodiment, an antibody of the invention can comprise a heavychain variable region comprising a VH CDR1 of SEQ ID NO: 147, a VH CDR2of SEQ ID NO: 148, and VH CDR3 of SEQ ID NO: 149, and can furthercomprise a light chain variable region comprising a VL CDR1 of SEQ IDNO:152, a VL CDR2 of SEQ ID NO: 153, and a VL CDR3 of SEQ ID NO: 154.

In one embodiment, an antibody of the invention can comprise a heavychain variable region comprising a VH CDR1 of SEQ ID NO: 147, a VH CDR2of SEQ ID NO: 148, and VH CDR3 of SEQ ID NO: 149, and can furthercomprise a light chain variable region comprising a VL CDR1 of SEQ IDNO:326, a VL CDR2 of SEQ ID NO: 327, and a VL CDR3 of SEQ ID NO: 328.

In one embodiment, an antibody of the invention can comprise a heavychain variable region comprising a VH CDR1 of SEQ ID NO: 156, a VH CDR2of SEQ ID NO: 157, and VH CDR3 of SEQ ID NO: 158, and can furthercomprise a light chain variable region comprising a VL CDR1 of SEQ IDNO:160, a VL CDR2 of SEQ ID NO: 161, and a VL CDR3 of SEQ ID NO: 162.

In one embodiment, an antibody of the invention can comprise a heavychain variable region comprising a VH CDR1 of SEQ ID NO: 164, a VH CDR2of SEQ ID NO: 165, and VH CDR3 of SEQ ID NO: 166, and can furthercomprise a light chain variable region comprising a VL CDR1 of SEQ IDNO:168, a VL CDR2 of SEQ ID NO: 169, and a VL CDR3 of SEQ ID NO: 170.

In one embodiment, an antibody of the invention can comprise a heavychain variable region comprising a VH CDR1 of SEQ ID NO: 172, a VH CDR2of SEQ ID NO: 173, and VH CDR3 of SEQ ID NO: 174, and can furthercomprise a light chain variable region comprising a VL CDR1 of SEQ IDNO:176, a VL CDR2 of SEQ ID NO: 177, and a VL CDR3 of SEQ ID NO: 178.

In one embodiment, an antibody of the invention can comprise a heavychain variable region comprising a VH CDR1 of SEQ ID NO: 180, a VH CDR2of SEQ ID NO: 181, and VH CDR3 of SEQ ID NO: 182, and can furthercomprise a light chain variable region comprising a VL CDR1 of SEQ IDNO:184, a VL CDR2 of SEQ ID NO: 185, and a VL CDR3 of SEQ ID NO: 186.

In one embodiment, an antibody of the invention can comprise a heavychain variable region comprising a VH CDR1 of SEQ ID NO: 188, a VH CDR2of SEQ ID NO: 189, and VH CDR3 of SEQ ID NO: 190, and can furthercomprise a light chain variable region comprising a VL CDR1 of SEQ IDNO:192, a VL CDR2 of SEQ ID NO: 193, and a VL CDR3 of SEQ ID NO: 194.

In one embodiment, an antibody of the invention can comprise a heavychain variable region comprising a VH CDR1 of SEQ ID NO: 196, a VH CDR2of SEQ ID NO: 197, and VH CDR3 of SEQ ID NO: 198, and can furthercomprise a light chain variable region comprising a VL CDR1 of SEQ IDNO:200, a VL CDR2 of SEQ ID NO: 201, and a VL CDR3 of SEQ ID NO: 202.

In one embodiment, an antibody of the invention can comprise a heavychain variable region comprising a VH CDR1 of SEQ ID NO: 204, a VH CDR2of SEQ ID NO: 205, and VH CDR3 of SEQ ID NO: 206, and can furthercomprise a light chain variable region comprising a VL CDR1 of SEQ IDNO:208, a VL CDR2 of SEQ ID NO: 209, and a VL CDR3 of SEQ ID NO: 210.

In one embodiment, an antibody of the invention can comprise a heavychain variable region comprising a VH CDR1 of SEQ ID NO: 212, a VH CDR2of SEQ ID NO: 213, and VH CDR3 of SEQ ID NO: 214, and can furthercomprise a light chain variable region comprising a VL CDR1 of SEQ IDNO:216, a VL CDR2 of SEQ ID NO: 217, and a VL CDR3 of SEQ ID NO: 218.

In one embodiment, an antibody of the invention can comprise a heavychain variable region comprising a VH CDR1 of SEQ ID NO: 220, a VH CDR2of SEQ ID NO: 221, and VH CDR3 of SEQ ID NO: 222, and can furthercomprise a light chain variable region comprising a VL CDR1 of SEQ IDNO:224, a VL CDR2 of SEQ ID NO: 225, and a VL CDR3 of SEQ ID NO: 226.

In one embodiment, an antibody of the invention can comprise a heavychain variable region comprising a VH CDR1 of SEQ ID NO: 228, a VH CDR2of SEQ ID NO: 229, and VH CDR3 of SEQ ID NO: 230, and can furthercomprise a light chain variable region comprising a VL CDR1 of SEQ IDNO:232, a VL CDR2 of SEQ ID NO: 233, and a VL CDR3 of SEQ ID NO: 234.

In one embodiment, an antibody of the invention can comprise a heavychain variable region comprising a VH CDR1 of SEQ ID NO: 236, a VH CDR2of SEQ ID NO: 237, and VH CDR3 of SEQ ID NO: 238, and can furthercomprise a light chain variable region comprising a VL CDR1 of SEQ IDNO:240, a VL CDR2 of SEQ ID NO: 241, and a VL CDR3 of SEQ ID NO: 242.

In one embodiment, an antibody of the invention can comprise a heavychain variable region comprising a VH CDR1 of SEQ ID NO: 244, a VH CDR2of SEQ ID NO: 245, and VH CDR3 of SEQ ID NO: 246, and can furthercomprise a light chain variable region comprising a VL CDR1 of SEQ IDNO:248, a VL CDR2 of SEQ ID NO: 249, and a VL CDR3 of SEQ ID NO: 250.

In one embodiment, an antibody of the invention can comprise a heavychain variable region comprising a VH CDR1 of SEQ ID NO: 252, a VH CDR2of SEQ ID NO: 253, and VH CDR3 of SEQ ID NO: 254, and can furthercomprise a light chain variable region comprising a VL CDR1 of SEQ IDNO:256, a VL CDR2 of SEQ ID NO: 257, and a VL CDR3 of SEQ ID NO: 258.

In one embodiment, an antibody of the invention can comprise a heavychain variable region comprising a VH CDR1 of SEQ ID NO: 260, a VH CDR2of SEQ ID NO: 261, and VH CDR3 of SEQ ID NO: 262, and can furthercomprise a light chain variable region comprising a VL CDR1 of SEQ IDNO:264, a VL CDR2 of SEQ ID NO: 265, and a VL CDR3 of SEQ ID NO: 266.

In one embodiment, an antibody of the invention can comprise a heavychain variable region comprising a VH CDR1 of SEQ ID NO: 268, a VH CDR2of SEQ ID NO: 269, and VH CDR3 of SEQ ID NO: 270, and can furthercomprise a light chain variable region comprising a VL CDR1 of SEQ IDNO:272, a VL CDR2 of SEQ ID NO: 273, and a VL CDR3 of SEQ ID NO: 274.

In one embodiment, an antibody of the invention is an antibodycomprising an amino acid sequence of the V_(H) and/or V_(L) region asdepicted in FIGS. 1A-1K and 3A-3R.

In another embodiment, an antibody of the invention is characterized bythe preservation of the cognate pairing of the heavy and light chainthat is present in a human B-cell.

In one embodiment, an antibody of the invention comprises a heavy chainvariable region (VH) comprising, or consisting of an amino acid sequenceselected from the group consisting of SEQ ID NO: 1, 10, 18, 26, 35, 45,53, 61, 69, 77, 87, 130, 138, 146, 155, 163, 171, 179, 187, 195, 203,211, 219, 227, 235, 243, 251, 259, and 267. In one embodiment, anantibody of the invention comprises a light chain variable region (VL)comprising, or consisting of an amino acid sequence selected from thegroup consisting of SEQ ID NO: 6, 14, 22, 31, 40, 49, 57, 65, 73, 82,122, 134, 142, 150, 151, 159, 167, 175, 183, 191, 199, 207, 215, 223,231, 239, 247, 255, 263, and 271. In one embodiment, an antibody of theinvention comprises a heavy chain variable region (VH) comprising, orconsisting of an amino acid sequence selected from the group consistingof SEQ ID NO: 1, 10, 18, 26, 35, 45, 53, 61, 69, 77, 87, 130, 138, 146,155, 163, 171, 179, 187, 195, 203, 211, 219, 227, 235, 243, 251, 259,and 267, and further comprises a light chain variable region (VL)comprising, or consisting of an amino acid sequence selected from thegroup consisting of SEQ ID NO: 6, 14, 22, 31, 40, 49, 57, 65, 73, 82,122, 134, 142, 150, 151, 159, 167, 175, 183, 191, 199, 207, 215, 223,231, 239, 247, 255, 263, and 271. In a specific embodiment, the antibodycomprises a VH of SEQ ID NO: 1 and a VL of SEQ ID NO: 6; or a VH of SEQID NO: 10 and a VL of SEQ ID NO: 14; or a VH of SEQ ID NO: 18 and a VLof SEQ ID NO: 22; or a VH of SEQ ID NO: 26 and a VL of SEQ ID NO: 31; ora VH of SEQ ID NO: 35 and a VL of SEQ ID NO: 40, or a VH of SEQ ID NO:45and a VL of SEQ ID NO: 49; or a VH of SEQ ID NO: 53 and a VL of SEQ IDNO: 57; or a VH of SEQ ID NO: 61 and a VL of SEQ ID NO: 65; or a VH ofSEQ ID NO: 69 and a VL of SEQ ID NO: 73; or a VH of SEQ ID NO: 77 and aVL of SEQ ID NO: 82, or a VH of SEQ ID NO:87 and a VL of SEQ ID NO: 122,or a VH of SEQ ID NO:130 and a VL of SEQ ID NO: 134, or a VH of SEQ IDNO:138 and a VL of SEQ ID NO: 142, or a VH of SEQ ID NO:146 and a VL ofSEQ ID NO: 150, or a VH of SEQ ID NO:146 and a VL of SEQ ID NO: 151, ora VH of SEQ ID NO:155 and a VL of SEQ ID NO: 159, or a VH of SEQ IDNO:163 and a VL of SEQ ID NO: 167, or a VH of SEQ ID NO:171 and a VL ofSEQ ID NO: 175, or a VH of SEQ ID NO:179 and a VL of SEQ ID NO: 183, ora VH of SEQ ID NO:187 and a VL of SEQ ID NO: 191, or a VH of SEQ IDNO:195 and a VL of SEQ ID NO: 199, or a VH of SEQ ID NO:203 and a VL ofSEQ ID NO: 207, or a VH of SEQ ID NO:211 and a VL of SEQ ID NO: 215, ora VH of SEQ ID NO:219 and a VL of SEQ ID NO: 223, or a VH of SEQ IDNO:227 and a VL of SEQ ID NO: 231, or a VH of SEQ ID NO:235 and a VL ofSEQ ID NO: 239, or a VH of SEQ ID NO:243 and a VL of SEQ ID NO: 247, ora VH of SEQ ID NO:251 and a VL of SEQ ID NO: 255, or a VH of SEQ IDNO:259 and a VL of SEQ ID NO: 263, or a VH of SEQ ID NO:267 and a VL ofSEQ ID NO: 271.

Alternatively, the TDP-binding molecule of the invention is apolypeptide such as an antibody (including an antigen-binding fragmentof an antibody, or a derivative or variant thereof), which competes forbinding to TDP-43, with at least one antibody having a V_(H) and/orV_(L) region as depicted in FIGS. 1A-1K and 3A-3R. Those antibodies canbe human as well, in particular for therapeutic applications.Alternatively, the antibody is a murine, murinized or chimericmurine-human antibody, which is particularly useful for diagnosticmethods and efficacy and safety studies in animals.

As discussed herein, the TDP-43 epitope of a completely human antibodyis particularly relevant for diagnostic and therapeutic applications dueto the fact that the antibody was initially generation as a result of ahuman immune response. Therefore, human completely human monoclonalantibodies of the invention recognize epitopes which are of particularphysiological relevance and which might not be accessible or lessimmunogenic using conventional immunization and other antibody screeningprocesses for the generation of for example, mouse monoclonal antibodiesand antibodies derived from in vitro screening of phage displaylibraries. Therefore, the invention also extends generally toanti-TDP-43 antibodies and other TDP-43 binding molecules which competewith a human monoclonal antibody of the invention for specific bindingto TDP-43. According to one embodiment, the antibody, or other TDP-43binding molecule, competes with an antibody containing the variabledomains disclosed in FIGS. 1A-1K and 3A-3R for binding with TDP-43. Inanother embodiment, the antibody or other TDP-43 binding moleculecompetes with a reference antibody selected from the group consistingof: NI-205.3F10, NI-205.51C1, NI-205.21G2, NI-205.8A2, NI-205.15F12,NI-205.113C4, NI-205.25F3, NI-205.87E7, NI-205.21G1, NI-205.68G5,NI-205.20A1, NI205.41D1, NI205.29E11, NI205.9E12, NI205.98H6,NI205.10D3, NI205.44B2, NI205.38H2, NI205.36D5, NI205.58E11, NI205.14H5,NI205.31D2, NI205.8F8, NI205.31C11, NI205.8C10, NI205.10H7, NI205.1A9,NI205.14W3, and NI205.19G5, for binding with TDP-43.

The invention also encompasses anti-TDP-43 antibodies and other TDP-43binding molecules which bind to the same epitope of TDP-43 as a humanmonoclonal antibody of the invention. According to one embodiment, theantibody (including TDP-43 binding antibody fragments and variant orderivative thereof) or other TDP-43 binding molecule binds to the sameepitope of TDP-43 as an antibody containing the variable domainsdisclosed in FIGS. 1A-1K and 3A-3R. In another embodiment, the antibody(including TDP-43 binding antibody fragments and variant or derivativethereof) or other TDP-43 binding molecule binds to the same epitope ofTDP-43 as a reference antibody selected from the group consisting of:NI-205.3F10, NI-205.51C1, NI-205.21G2, NI-205.8A2, NI-205.15F12,NI-205.113C4, NI-205.25F3, NI-205.87E7, NI-205.21G1, NI-205.68G5,NI-205.20A1, NI205.41D1, NI205.29E11, NI205.9E12, NI205.98H6,NI205.10D3, NI205.44B2, NI205.38H2, NI205.36D5, NI205.58E11, NI205.14H5,NI205.31D2, NI205.8F8, NI205.31C11, NI205.8C10, NI205.10H7, NI205.1A9,NI205.14W3, and NI205.19G5.

In another embodiment, the invention encompasses an antibody (includingan antigen-binding fragment, variant or derivatives thereof), thatspecifically binds to a TDP-43 polypeptide sequence selected from:QYGDVMDVFIP (SEQ ID NO: 123); AAIGWGSASNA (SEQ ID NO: 124); DMTEDELREFF(SEQ ID NO: 125), EDENDEP (SEQ ID NO: 126), VQVKKDL (SEQ ID NO: 127),KEYFSTF (SEQ ID NO: 128), IIKGISV (SEQ ID NO:315), NQSGPSG (SEQ IDNO:316), FNGGFGS (SEQ ID NO:317), FGNSRGGGAGL (SEQ ID NO:318),SNAGSGSGFNG (SEQ ID NO:319), QLERSGRFGGN (SEQ ID NO:320), EIPSEDD (SEQID NO:321), FNGGFGS SMDS (SEQ ID NO:322) and SINPAMMAAAQAALQSSWGMMGMLASQ(SEQ ID NO:323). In another embodiment, the invention encompasses anantibody (including an antigen-binding fragment, variant or derivativesthereof), that specifically binds to TDP-43 polypeptides FGNSRGGGAGL(SEQ ID NO:318) and SNAGSGSGFNG (SEQ ID NO:319). In another embodiment,the invention encompasses an antibody (including an antigen-bindingfragment, variant or derivatives thereof), that specifically binds toTDP-43 polypeptide SINPAMMAAAQAALQSSWGMMGMLASQ (SEQ ID NO:323), but doesnot specifically bind to SINPGGGAAAQAALQSSWGMMGMLASQ (SEQ ID NO:314).

Competition between antibodies is determined by an assay in which theimmunoglobulin under test inhibits specific binding of a referenceantibody to a common antigen, such as TDP-43. Numerous types ofcompetitive binding assays are known in the art and can routinely beapplied or modified to test the ability of two compounds to compete forbinding to an antigen, such as, solid phase direct or indirectradioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay; see Stahli et al.,Methods in Enzymology 9 (1983), 242-253; solid phase directbiotin-avidin EIA; see Kirkland et al., J. Immunol. 137 (1986),3614-3619, and Cheung et al., Virology 176 (1990), 546-552; solid phasedirect labeled assay, solid phase direct labeled sandwich assay; seeHarlow and Lane, Antibodies, A Laboratory Manual, Cold Spring HarborPress (1988); solid phase direct label RIA using I¹²⁵ label; see Morelet al, Molec. Immunol. 25 (1988), 7-15 and Moldenhauer et al., Scand. J.Immunol. 32 (1990), 77-82. Typically, such an assay involves the use ofpurified TDP-43 or aggregates thereof bound to a solid surface or cellsbearing either of these, an unlabeled test immunoglobulin and a labeledreference immunoglobulin, e.g., a human monoclonal antibody of theinvention. Competitive inhibition is measured by determining the amountof label bound to the solid surface or cells in the presence of the testimmunoglobulin. Usually the test immunoglobulin is present in excess. Inone embodiment, the competitive binding assay is performed underconditions as described for the ELISA assay in the appended Examples.Antibodies identified by competition assay (competing antibodies)include antibodies binding to the same epitope as the reference antibodyand antibodies binding to an adjacent epitope sufficiently proximal tothe epitope bound by the reference antibody for steric hindrance tooccur. Usually, when a competing antibody is present in excess, it willinhibit specific binding of a reference antibody to a common antigen byat least 50% or 75%. Hence, the invention is further drawn to anantibody (e.g., an antigen-binding fragment of an antibody), where theantibody competitively inhibits binding to TDP-43 by a referenceantibody selected from the group consisting of NI-205.3F10, NI-205.51C1,NI-205.21G2, NI-205.8A2, NI-205.15F12, NI-205.113C4, NI-205.25F3,NI-205.87E7, NI-205.21G1, NI-205.68G5, NI-205.20A1, NI205.41D1,NI205.29E11, NI205.9E12, NI205.98H6, NI205.10D3, NI205.44B2, NI205.38H2,NI205.36D5, NI205.58E11, NI205.14H5, NI205.31D2, NI205.8F8, NI205.31C11,NI205.8C10, NI205.10H7, NI205.1A9, NI205.14W3, and NI205.19G5.

The invention also provides antibodies that comprise, consistessentially of, or consist of, variants (including derivatives) of theantibody molecules (e.g., the V_(H) regions and/or V_(L) regions)described herein, which antibodies immunospecifically bind to a TDP-43polypeptide or fragment or variant thereof. Standard techniques known inthe art can be used to introduce mutations in the nucleotide sequenceencoding a molecule of the invention, including, for example,site-directed mutagenesis and PCR-mediated mutagenesis which result inamino acid substitutions. Preferably, the variants (includingderivatives) encode less than 50 amino acid substitutions, less than 40amino acid substitutions, less than 30 amino acid substitutions, lessthan 25 amino acid substitutions, less than 20 amino acid substitutions,less than 15 amino acid substitutions, less than 10 amino acidsubstitutions, less than 5 amino acid substitutions, less than 4 aminoacid substitutions, less than 3 amino acid substitutions, or less than 2amino acid substitutions relative to the reference V_(H) region,V_(H)CDR1, V_(H)CDR2, V_(H)CDR3, VL region, V_(L)CDR1, V_(L)CDR2, orV_(L)CDR3.

According to one embodiment, the invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (VII), where at least one ofV_(H)-CDRs of the heavy chain variable region or at least two of theV_(H)-CDRs of the heavy chain variable region are at least 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% identical to reference heavy chainV_(H)-CDR1, V_(H)-CDR2 or V_(H)-CDR3 amino acid sequences from theantibodies disclosed herein. Alternatively, the V_(H)-CDR1, V_(H)-CDR2and V_(H)-CDR3 regions of the V_(H) are at least 80%, 85%, 90%, 95%,96%, 97%, 98% or 99% identical to reference heavy chain V_(H)-CDR1,V_(H)-CDR2 and V_(H)-CDR3 amino acid sequences from the antibodiesdisclosed herein. Thus, according to this embodiment a heavy chainvariable region of the invention has V_(H)-CDR1, V_(H)-CDR2 andV_(H)-CDR3 polypeptide sequences related to the groups shown in FIGS.1A-1K and 3A-3R. While FIGS. 1A-1K and 3A-3R shows V_(H)-CDRs defined bythe Kabat system, other CDR definitions, e.g., V_(H)-CDRs defined by theChothia system, are also included in the invention, and can be easilyidentified by a person of ordinary skill in the art using the datapresented in FIGS. 1A-1K and 3A-3R. In one embodiment, the amino acidsequence of the reference VH CDR1 is SEQ ID NO: 3, 11, 19, 28, 37, 46,54, 62, 70, 79, 88, 131, 139, 147, 156, 164, 172, 180, 188, 196, 204,212, 220, 228, 236, 244, 252, 260, or 268; the amino acid sequence ofthe reference VH CDR2 is SEQ ID NO: 4, 12, 20, 29, 38, 47, 55, 63, 71,80, 89, 132, 140, 148, 157, 165, 173, 181, 189, 197, 205, 213, 221, 229,237, 245, 253, 261, or 269; and the amino acid sequence of the referenceVH CDR3 is SEQ ID NO: 5, 13, 21, 30, 39, 48, 56, 64, 72, 81, 90, 133,141, 149, 158, 166, 174, 182, 190, 198, 206, 214, 222, 230, 238, 246,254, 262, or 270.

In another embodiment, the invention provides an isolated polypeptidecomprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (V_(H)) in which theV_(H)-CDR1, V_(H)-CDR2 and V_(H)-CDR3 regions have polypeptide sequenceswhich are identical to the V_(H)-CDR1, V_(H)-CDR2 and V_(H)-CDR3 groupsshown in FIGS. 1A-1K and 3A-3R. In one embodiment, the amino acidsequence of the VH CDR1 is SEQ ID NO: 3, 11, 19, 28, 37, 46, 54, 62, 70,79, 88, 131, 139, 147, 156, 164, 172, 180, 188, 196, 204, 212, 220, 228,236, 244, 252, 260, or 268; the amino acid sequence of the reference VHCDR2 is SEQ ID NO: 4, 12, 20, 29, 38, 47, 55, 63, 71, 80, 89, 132, 140,148, 157, 165, 173, 181, 189, 197, 205, 213, 221, 229, 237, 245, 253,261, or 269; and the amino acid sequence of the reference VH CDR3 is SEQID NO: 5, 13, 21, 30, 39, 48, 56, 64, 72, 81, 90, 133, 141, 149, 158,166, 174, 182, 190, 198, 206, 214, 222, 230, 238, 246, 254, 262, or 270.

In another embodiment, the invention provides an isolated polypeptidecomprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (V_(H)) in which theV_(H)-CDR1, V_(H)-CDR2 and V_(H)-CDR3 regions have polypeptide sequenceswhich are identical to the V_(H)-CDR1, V_(H)-CDR2 and V_(H)-CDR3 groupsshown in FIGS. 1A-1K and 3A-3R, except for one, two, three, four, five,six, seven, eight, nine, or ten amino acid substitutions in any oneV_(H)-CDR. In certain embodiments the amino acid substitutions areconservative. In one embodiment, the amino acid sequence of the VH CDR1is SEQ ID NO: 3, 11, 19, 28, 37, 46, 54, 62, 70, 79, 88, 131, 139, 147,156, 164, 172, 180, 188, 196, 204, 212, 220, 228, 236, 244, 252, 260, or268; the amino acid sequence of the reference VH CDR2 is SEQ ID NO: 4,12, 20, 29, 38, 47, 55, 63, 71, 80, 89, 132, 140, 148, 157, 165, 173,181, 189, 197, 205, 213, 221, 229, 237, 245, 253, 261, or 269; and theamino acid sequence of the reference VH CDR3 is SEQ ID NO: 5, 13, 21,30, 39, 48, 56, 64, 72, 81, 90, 133, 141, 149, 158, 166, 174, 182, 190,198, 206, 214, 222, 230, 238, 246, 254, 262, or 270.

In another embodiment, the invention provides an isolated polypeptidecomprising, consisting essentially of, or consisting of animmunoglobulin light chain variable region (V_(L)), where at least oneof the V_(L)-CDRs of the light chain variable region or at least two ofthe V_(L)-CDRs of the light chain variable region are at least 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% identical to reference light chainV_(L)-CDR1, V_(L)-CDR2 or V_(L)-CDR3 amino acid sequences fromantibodies disclosed herein. Alternatively, the V_(L)-CDR1, V_(L)-CDR2and V_(L)-CDR3 regions of the VL are at least 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% identical to reference light chain V_(L)-CDR1,V_(L)-CDR2 and V_(L)-CDR3 amino acid sequences from antibodies disclosedherein. Thus, according to this embodiment a light chain variable regionof the invention has V_(L)-CDR1, V_(L)-CDR2 and V_(L)-CDR3 polypeptidesequences related to the polypeptides shown in FIGS. 1A-1K and 3A-3R.While FIGS. 1A-1K and 3A-3R show V_(L)-CDRs defined by the Kabat system,other CDR definitions, e.g., V_(L)-CDRs defined by the Chothia system,are also included in the invention. In one embodiment, the amino acidsequence of the reference VL CDR1 is SEQ ID NO: 7, 15, 23, 32, 42, 50,58, 66, 74, 84, 91, 135, 143, 152, 160, 168, 176, 184, 192, 200, 208,216, 224, 232, 240, 248, 256, 264, 272 or 326; the amino acid sequenceof the reference VL CDR2 is SEQ ID NO: 8, 16, 24, 33, 43, 51, 59, 67,75, 85, 92, 136, 144, 153, 161, 169, 177, 185, 193, 201, 209, 217, 225,233, 241, 249, 257, 265, 273 or 327; and the amino acid sequence of thereference VL CDR3 is SEQ ID NO: 9, 17, 25, 34, 44, 52, 60, 68, 76, 86,93, 137, 145, 154, 162, 170, 178, 186, 194, 202, 210, 218, 226, 234,242, 250, 258, 266, 274 or 328.

In another embodiment, the invention provides an isolated polypeptidecomprising, consisting essentially of, or consisting of animmunoglobulin light chain variable region (V_(L)) in which theV_(L)-CDR1, V_(L)-CDR2 and V_(L)-CDR3 regions have polypeptide sequenceswhich are identical to the V_(L)-CDR1, V_(L)-CDR2 and V_(L)-CDR3 groupsshown in FIGS. 1A-1K and 3A-3R. In one embodiment, the amino acidsequence of the VL CDR1 is SEQ ID NO: 7, 15, 23, 32, 42, 50, 58, 66, 74,84, 91, 135, 143, 152, 160, 168, 176, 184, 192, 200, 208, 216, 224, 232,240, 248, 256, 264, 272 or 326; the amino acid sequence of the VL CDR2is SEQ ID NO: 8, 16, 24, 33, 43, 51, 59, 67, 75, 85, 92, 136, 144, 153,161, 169, 177, 185, 193, 201, 209, 217, 225, 233, 241, 249, 257, 265,273 or 327; and the amino acid sequence of the VL CDR3 is SEQ ID NO: 9,17, 25, 34, 44, 52, 60, 68, 76, 86, 93, 137, 145, 154, 162, 170, 178,186, 194, 202, 210, 218, 226, 234, 242, 250, 258, 266, 274 or 328.

In another embodiment, the invention provides an isolated polypeptidecomprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (VH) in which the V_(H)-CDR1,V_(H)-CDR2 and V_(H)-CDR3 regions have polypeptide sequences which areidentical to the V_(H)-CDR1, V_(H)-CDR2 and V_(H)-CDR3 groups shown inFIGS. 1A-1K and 3A-3R, except for one, two, three, four, five, six,seven, eight, nine, or ten amino acid substitutions in any oneV_(L)-CDR. In certain embodiments the amino acid substitutions areconservative. In one embodiment, the amino acid sequence of the VL CDR1is SEQ ID NO: 7, 15, 23, 32, 42, 50, 58, 66, 74, 84, 91, 135, 143, 152,160, 168, 176, 184, 192, 200, 208, 216, 224, 232, 240, 248, 256, 264,272 or 326; the amino acid sequence of the VL CDR2 is SEQ ID NO: 8, 16,24, 33, 43, 51, 59, 67, 75, 85, 92, 136, 144, 153, 161, 169, 177, 185,193, 201, 209, 217, 225, 233, 241, 249, 257, 265, 273 or 327; and theamino acid sequence of the VL CDR3 is SEQ ID NO: 9, 17, 25, 34, 44, 52,60, 68, 76, 86, 93, 137, 145, 154, 162, 170, 178, 186, 194, 202, 210,218, 226, 234, 242, 250, 258, 266, 274 or 328.

According to one embodiment, the invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (V_(H)) at least 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% identical to a reference heavy chainvariable region (V_(H)) amino acid sequence from the antibodiesdisclosed herein. Thus, according to this embodiment a heavy chainvariable region of the invention has a polypeptide sequence related tothe heavy chain variable regions shown in FIGS. 1A-1K and 3A-3R. In oneembodiment, the amino acid sequence of the reference heavy chainvariable region (V_(H)) is SEQ ID NO: 1, 10, 18, 26, 35, 45, 53, 61, 69,77, 87, 130, 138, 146, 155, 163, 171, 179, 187, 195, 203, 211, 219, 227,235, 243, 251, 259, or 267.

In another embodiment, the invention provides an isolated polypeptidecomprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (V_(H)) which is identical toa reference heavy chain variable region shown in FIGS. 1A-1K and 3A-3R.In one embodiment, the amino acid sequence of the reference heavy chainvariable region is SEQ ID NO: 1, 10, 18, 26, 35, 45, 53, 61, 69, 77, 87,130, 138, 146, 155, 163, 171, 179, 187, 195, 203, 211, 219, 227, 235,243, 251, 259, and 267.

In another embodiment, the invention provides an isolated polypeptidecomprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (V_(H)) having a polypeptidesequence which is identical to a reference heavy chain variable region(V_(H)) sequence shown in FIGS. 1A-1K and 3A-3R, except for one, two,three, four, five, six, seven, eight, nine, or ten amino acidsubstitutions. In certain embodiments the amino acid substitutions areconservative. In one embodiment, the amino acid sequence of thereference heavy chain variable region sequence is SEQ ID NO: 1, 10, 18,26, 35, 45, 53, 61, 69, 77, 87, 130, 138, 146, 155, 163, 171, 179, 187,195, 203, 211, 219, 227, 235, 243, 251, 259, or 267.

According to one embodiment, the invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin light chain variable region (V_(L)) at least 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% identical to a reference light chainvariable region (V_(L)) amino acid sequence from the antibodiesdisclosed herein. Thus, according to this embodiment a light chainvariable region of the invention has a polypeptide sequence related tothe light chain variable regions shown in FIGS. 1A-1K and 3A-3R. In oneembodiment, the amino acid sequence of the reference light chainvariable region (V_(L)) is SEQ ID NO: 6, 14, 22, 31, 40, 49, 57, 65, 73,82, 122, 134, 142, 150, 151, 159, 167, 175, 183, 191, 199, 207, 215,223, 231, 239, 247, 255, 263, and 271.

In another embodiment, the invention provides an isolated polypeptidecomprising, consisting essentially of, or consisting of animmunoglobulin light chain variable region (V_(L)) which is identical toa reference light chain variable region shown in FIGS. 1A-1K and 3A-3R.In one embodiment, the amino acid sequence of the reference light chainvariable region is SEQ ID NO: 6, 14, 22, 31, 40, 49, 57, 65, 73, 82,122, 134, 142, 150, 151, 159, 167, 175, 183, 191, 199, 207, 215, 223,231, 239, 247, 255, 263, and 271.

In another embodiment, the invention provides an isolated polypeptidecomprising, consisting essentially of, or consisting of animmunoglobulin light chain variable region (V_(L)) having a polypeptidesequence which is identical to a reference light chain variable region(V_(L)) sequence shown in FIGS. 1A-1K and 3A-3R, except for one, two,three, four, five, six, seven, eight, nine, or ten amino acidsubstitutions. In certain embodiments the amino acid substitutions areconservative. In one embodiment, the amino acid sequence of thereference light chain variable region sequence is SEQ ID NO: 6, 14, 22,31, 40, 49, 57, 65, 73, 82, 122, 134, 142, 150, 151, 159, 167, 175, 183,191, 199, 207, 215, 223, 231, 239, 247, 255, 263, and 271.

An immunoglobulin or its encoding nucleic acid (e.g., a cDNA) can befurther modified. Thus, in a further embodiment the method of theinvention comprises any one of the step(s) of producing a chimericantibody, murinized antibody, single-chain antibody, Fab-fragment,bi-specific antibody, fusion antibody, labeled antibody or an analog ofany one of those. Corresponding methods are known in the art and aredescribed, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual”,CSH Press, Cold Spring Harbor (1988). When derivatives of saidantibodies are obtained by for example, the phage display technique,surface plasmon resonance as employed in the BIAcore system can be usedto increase the efficiency of phage antibodies which bind to the sameepitope as that of any one of the antibodies described herein (Schier,Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol.Methods 183 (1995), 7-13). The production of chimeric antibodies isdescribed, for example, in International Application Publication No.WO89/09622. Methods for the production of humanized antibodies aredescribed in, e.g., European application EP-A1 0 239 400 andInternational Application WO90/07861. A further source of antibodies tobe utilized in accordance with the invention is so-called xenogeneicantibodies. The general principle for the production of xenogeneicantibodies such as human-like antibodies in mice is described in, e.g.,International Application Publication Nos. WO91/10741, WO94/02602,WO96/34096 and WO 96/33735. As discussed above, the antibody of theinvention can exist in a variety of forms besides complete antibodies;including, for example, Fv, Fab and F(ab)₂, as well as in single chains;see, e.g., International Application Publication No. WO88/09344.

The antibodies of the invention or their corresponding immunoglobulinchain(s) can be further modified using conventional techniques known inthe art, for example, by using amino acid deletion(s), insertion(s),substitution(s), addition(s), and/or recombination(s) and/or any othermodification(s) known in the art either alone or in combination. Methodsfor introducing such modifications in the nucleic acid sequence encodingthe amino acid sequence of an immunoglobulin chain are well known to aperson of ordinary skill in the art; see, e.g., Sambrook, MolecularCloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y.and Ausubel, Current Protocols in Molecular Biology, Green PublishingAssociates and Wiley Interscience, N.Y. (1994). Modifications of theantibody of the invention include chemical and/or enzymaticderivatizations at one or more constituent amino acids, including sidechain modifications, backbone modifications, and N- and C-terminalmodifications including acetylation, hydroxylation, methylation,amidation, and the attachment of carbohydrate or lipid moieties,cofactors, and the like. Likewise, the invention encompasses theproduction of chimeric proteins (i.e., fusion proteins) which comprisethe TDP-43 binding polypeptides of the invention such as antibodies, atthe amino terminus fused to heterologous molecule such as animmunostimulatory ligand at the carboxyl terminus; see, e.g.,International Application Publication No. WO00/30680 for correspondingtechnical details.

Additionally, the invention encompasses peptides and polypeptides thatspecifically bind TDP-43. For example containing the CDR3 region of thevariable region of any one of the mentioned antibodies, in particularCDR3 of the heavy chain since it has frequently been observed that heavychain CDR3 (HCDR3) is the region having a greater degree of variabilityand a predominant participation in antigen-antibody interaction. Suchpeptides and polypeptides can readily be synthesized or produced byrecombinant means to produce a TDP-43 binding molecule of the invention.Such methods are known to those of ordinary skill in the art. Peptidescan be synthesized for example, using automated peptide synthesizerswhich are commercially available. The peptides can also be produced byrecombinant techniques by incorporating the DNA expressing the peptideinto an expression vector and transforming cells with the expressionvector to produce the peptide.

Accordingly, the invention relates to TDP-43 binding molecules such as,antibodies (e.g., a TDP-43 binding fragment of an antibody) that displayone or more properties of the TDP-43 binding molecules described herein.For example, such antibodies and binding molecules can be tested fortheir binding specificity and affinity by for example, ELISA or WesternBlot and immunohistochemistry as described herein; see, e.g., theExamples. As disclosed in Example 2, the half maximal effectiveconcentration (EC₅₀) of NI-205.3F10, NI-205.51C1, NI-205.21G2,NI-205.8A2, NI-205.15F12, NI-205.113C4, NI-205.25F3, NI-205.87E7,NI-205.21G1, NI-205.68G5, NI-205.20A1, NI205.41D1, NI205.29E11,NI205.9E12, NI205.98H6, NI205.10D3, NI205.44B2, NI205.38H2, NI205.36D5,NI205.58E11, NI205.14H5, NI205.31D2, NI205.8F8, NI205.31C11, NI205.8C10,NI205.10H7, NI205.1A9, NI205.14W3, and NI205.19G5, was determined forhuman TDP-43 by direct ELISA to bind human TDP-43 with high affinity ata sub-mid nanomolar EC₅₀ (0.18-17.2 nM).

As an alternative to obtaining immunoglobulins directly from the cultureof immortalized B cells or B memory cells, the immortalized cells can beused as a source of rearranged heavy chain and light chain loci forsubsequent expression and/or genetic manipulation. Rearranged antibodygenes can be reverse transcribed from appropriate mRNAs to produce cDNA.If desired, the heavy chain constant region can be exchanged for that ofa different isotype or eliminated altogether. The variable regions canbe linked to encode single chain Fv regions. Multiple Fv regions can belinked to confer binding ability to more than one target or chimericheavy and light chain combinations can be employed. Once the geneticmaterial is available, design of analogs which retain the ability tobind a desired target is straightforward. Methods for cloning antibodyvariable regions and generation of recombinant antibodies are known inthe art and are described, for example, in Gilliland et al., TissueAntigens 47 (1996), 1-20; Doenecke et al., Leukemia 11 (1997),1787-1792.

Once the appropriate genetic material is obtained and, if desired,modified to encode an analog, the coding sequences, including those thatencode, at a minimum, the variable regions of the heavy and light chain,can be inserted into expression systems contained on vectors which canbe transfected into standard recombinant host cells. A variety of suchhost cells can be used; for efficient processing, however, mammaliancells can be considered. Mammalian cell lines useful for this purposeinclude, but are not limited to, CHO cells, HEK 293 cells, or NSO cells.

The production of the antibody or analog is then undertaken by culturingthe modified recombinant host under culture conditions appropriate forthe growth of the host cells and the expression of the coding sequences.The antibodies are then recovered by isolating them from the culture.The expression systems are designed to include signal peptides so thatthe resulting antibodies are secreted into the medium; however,intracellular production is also possible.

In accordance with the above, the invention also relates to apolynucleotide encoding the antibody or equivalent binding molecule ofthe invention. In one embodiment, the polynucleotide encodes at least avariable region of an immunoglobulin chain of the antibody describedabove. Typically, said variable region encoded by the polynucleotidecomprises at least one complementarity determining region (CDR) of theV_(H) and/or V_(L) of the variable region of said antibody.

The person of ordinary skill in the art will readily appreciate that thevariable domain of the antibody having the above-described variabledomain can be used for the construction of other polypeptides orantibodies of desired specificity and biological function. Thus, theinvention also encompasses polypeptides and antibodies comprising atleast one CDR of the above-described variable domain and whichadvantageously have substantially the same or similar binding propertiesas the antibody described in the appended examples. As generallyunderstood in the art, binding affinity can be enhanced by making aminoacid substitutions within the CDRs or within the hypervariable loops(Chothia and Lesk, J. Mol. Biol. 196 (1987), 901-917) which partiallyoverlap with the CDRs as defined by Kabat; see, e.g., Riechmann, et al,Nature 332 (1988), 323-327. Thus, the invention also relates toantibodies wherein one or more of the mentioned CDRs comprise one ormore, or not more than two amino acid substitutions. In one embodiment,the antibody of the invention comprises in one or both of itsimmunoglobulin chains two or all three CDRs of the variable regions asset forth in FIGS. 1A-1K and 3A-3R.

Binding molecules such as antibodies (including antigen-bindingfragments, variants, or derivatives thereof) of the invention, cancomprise a constant region which mediates one or more effectorfunctions. For example, binding of the C1 component of complement to anantibody constant region can activate the complement system. Activationof complement is important in the opsonization and lysis of cellpathogens. The activation of complement also stimulates the inflammatoryresponse and can also be involved in autoimmune hypersensitivity.Further, antibodies bind to receptors on various cells via the Fcregion, with a Fc receptor binding site on the antibody Fc regionbinding to a Fc receptor (FcR) on a cell. There are a number of Fcreceptors which are specific for different classes of antibody,including IgG (gamma receptors), IgE (epsilon receptors), IgA (alphareceptors) and IgM (mu receptors). Binding of antibody to Fc receptorson cell surfaces triggers a number of important and diverse biologicalresponses including engulfment and destruction of antibody-coatedparticles, clearance of immune complexes, lysis of antibody-coatedtarget cells by killer cells (called antibody-dependent cell-mediatedcytotoxicity, or ADCC), release of inflammatory mediators, placentaltransfer and control of immunoglobulin production.

Accordingly, certain embodiments of the invention include an antibody(including an antibody antigen-binding fragment, variant, or derivativethereof), in which at least a fraction of one or more of the constantregion domains has been substituted, deleted or otherwise altered so asto provide desired biochemical characteristics such as reduced effectorfunctions, the ability to non-covalently dimerize, increased ability tolocalize at the site of TDP-43 aggregation and deposition, reduced serumhalf-life, or increased serum half-life when compared with a whole,unaltered antibody of approximately the same immunogenicity. Forexample, certain antibodies for use in the diagnostic and treatmentmethods described herein are domain deleted antibodies which comprise apolypeptide chain similar to an immunoglobulin heavy chain, but whichlack at least a portion of one or more heavy chain domains. Forinstance, in certain embodiments an antibody of the invention is missingan entire domain of the constant region of the modified antibody, suchas, all or part of the CH2 domain. In other embodiments, antibodies ofthe invention useful for example in diagnostic or therapeutic methodshave a constant region, e.g., an IgG heavy chain constant region, whichis altered to eliminate glycosylation, referred to elsewhere herein asaglycosylated or “agly” antibodies. Such “agly” antibodies can beprepared enzymatically or by other techniques known in the art includingfor example, by engineering the consensus glycosylation site(s) in theconstant region. While not being bound by theory, it is believed that“agly” antibodies can have an improved safety and stability profile invivo. Methods of producing aglycosylated antibodies, having desiredeffector function are found for example in International ApplicationPublication No. WO2005/018572, which is herein incorporated by referencein its entirety.

In certain antibodies, including antigen-binding antibody fragments andvariants described herein, the Fc portion can be mutated to decreaseeffector function using techniques known in the art. For example, thedeletion or inactivation (through point mutations or other means) of aconstant region domain can reduce Fc receptor binding of the circulatingmodified antibody thereby increasing TDP-43 localization. In other casesit can be that constant region modifications consistent with the instantinvention moderate complement binding and thus reduce the serumhalf-life and nonspecific association of a conjugated cytotoxin. Yetother modifications of the constant region can be used to modifydisulfide linkages or oligosaccharide moieties that allow for enhancedlocalization due to increased antigen specificity or antibodyflexibility. The resulting physiological profile, bioavailability andother biochemical effects of the modifications, such as TDP-43localization, biodistribution and serum half-life, can routinely bemeasured using techniques known in the art.

In certain embodiments, the Fc portion of antibodies of the inventionare mutated or exchanged for alternative protein sequences to increasethe cellular uptake of antibodies by way of example, by enhancingreceptor-mediated endocytosis of antibodies via Fcγ receptors, LRP, orThy1 receptors or by ‘SuperAntibody Technology’, which is said to enableantibodies to be shuttled into living cells without harming them(Muller, S., et al., Expert Opin. Biol. Ther. (2005), 237-241). Forexample, the generation of fusion proteins of the antibody bindingregion and the cognate protein ligands of cell surface receptors orbispecific or multi-specific antibodies with a specific sequencesbinding to TDP-43 as well as a cell surface receptor can be engineeredusing techniques known in the art.

In certain antibodies, including antigen-binding antibody fragments andvariants described herein, the Fc portion can be mutated or exchangedfor alternative protein sequences or the antibody can be chemicallymodified to increase its blood brain barrier penetration.

Modified forms of antibodies (e.g., antigen-binding fragments ofantibodies and variants, or derivatives thereof) of the invention can bemade from whole precursor or parent antibodies using techniques known inthe art. Exemplary techniques are discussed in more detail herein.Antibodies of the invention, including antigen-binding antibodyfragments and variants described herein, can routinely be made ormanufactured using techniques known in the art. In certain embodiments,antibodies (including antibody fragments and derivatives) are“recombinantly produced,” i.e., are produced using recombinant DNAtechnology. Exemplary techniques for making antibodies are discussed inmore detail elsewhere herein.

Antibodies (including antigen-binding fragments of antibodies andvariants, and derivatives thereof) of the invention also includederivatives that are modified, e.g., by the covalent attachment of anytype of molecule to the antibody such that the covalent attachment doesnot prevent the antibody from specifically binding to its cognateepitope. For example, but not by way of limitation, the antibodyderivatives include antibodies that have been modified, e.g., byglycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Any ofnumerous chemical modifications can be carried out by known techniques,including, but not limited to specific chemical cleavage, acetylation,formylation, metabolic synthesis of tunicamycin, etc. Additionally, thederivative can contain one or more non-classical amino acids.

In particular embodiments the TDP-43 binding molecules of the invention,are polypeptides such as antibodies (including antigen-bindingfragments, variants, or derivatives thereof) do not elicit a deleteriousimmune response in the animal to be treated, e.g., in a human. Incertain embodiments, binding molecules, e.g., antibodies (includingantigen-binding fragments of antibodies) of the invention are derivedfrom a patient, e.g., a human patient, and are subsequently used in thesame species from which they are derived, e.g., human, thus, alleviatingor minimizing the occurrence of deleterious immune responses.

De-immunization can also be used to decrease the immunogenicity of anantibody. As used herein, the term “de-immunization” includes alterationof an antibody to modify T cell epitopes; see, e.g., InternationalApplication Publication Nos. WO98/52976 and WO00/34317. For example,V_(H) and V_(L) sequences from the starting antibody are analyzed and ahuman T cell epitope “map” from each V region showing the location ofepitopes in relation to complementarity determining regions (CDRs) andother key residues within the sequence. Individual T cell epitopes fromthe T cell epitope map are analyzed in order to identify alternativeamino acid substitutions with a low risk of altering activity of thefinal antibody. A range of alternative V_(H) and V_(L) sequences aredesigned comprising combinations of amino acid substitutions and thesesequences are subsequently incorporated into a range of bindingpolypeptides, e.g., TDP-43-specific antibodies, including immunospecificfragments thereof, for use in the diagnostic and treatment methodsdisclosed herein, which are then tested for function. Typically, between12 and 24 variant antibodies are generated and tested. Complete heavyand light chain genes comprising modified V and human C regions are thencloned into expression vectors and the subsequent plasmids introducedinto cell lines for the production of whole antibody. The antibodies arethen compared in appropriate biochemical and biological assays, and theoptimal variant is identified.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed.(1988); Hammerling et al., in: Monoclonal Antibodies and T-CellHybridomas Elsevier, N.Y., 563-681 (1981), each of which references areherein incorporated by reference in their entireties. The term“monoclonal antibody” as used herein is not limited to antibodiesproduced through hybridoma technology. The term “monoclonal antibody”refers to an antibody that is derived from a single isolated clone,including any eukaryotic, prokaryotic, or phage clone, and not themethod by which it is produced. Thus, the term “monoclonal antibody” isnot limited to antibodies produced through hybridoma technology. Incertain embodiments, antibodies of the invention are derived from humanB cells which have been immortalized via transformation withEpstein-Barr virus, as described herein. For clarity, the term“monoclonal antibody” as used herein, does not encompass an endogenousantibody that can be isolated from the plasma of a host organism.

In the well known hybridoma process (Kohler et al., Nature 256 (1975),495) the relatively short-lived, or mortal, lymphocytes from a mammal,e.g., B cells derived from a human subject as described herein, arefused with an immortal tumor cell line (e.g., a myeloma cell line),thus, producing hybrid cells or “hybridomas” which are both immortal andcapable of producing the genetically coded antibody of the B cell. Theresulting hybrids are segregated into single genetic strains byselection, dilution, and re-growth with each individual straincomprising specific genes for the formation of a single antibody. Theselected hybridomas produce antibodies, which are homogeneous against adesired antigen and, in reference to their pure genetic parentage, aretermed “monoclonal”.

Hybridoma cells thus prepared are seeded and grown in a suitable culturemedium that contain one or more substances that inhibit the growth orsurvival of the unfused, parental myeloma cells. Reagents, cell linesand methods for forming, selecting and growing of hybridomas are knownin the art. Generally, culture medium in which the hybridoma cells aregrowing is assayed for production of monoclonal antibodies against thedesired antigen. The binding specificity of the monoclonal antibodiesproduced by hybridoma cells is determined by in vitro assays such asimmunoprecipitation, radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA) as described herein. After hybridoma cellsare identified that produce antibodies of the desired specificity,affinity and/or activity, the clones can be subcloned by limitingdilution procedures and grown by standard methods; see, e.g., Goding,Monoclonal Antibodies: Principles and Practice, Academic Press, pp.59-103 (1986). It will further be appreciated that the monoclonalantibodies secreted by the hybridoma subclones can be separated fromculture medium, ascites fluid or serum by conventional purificationprocedures such as, for example, protein-A, hydroxylapatitechromatography, gel electrophoresis, dialysis or affinitychromatography.

In another embodiment, lymphocytes are selected by micromanipulation andthe variable genes isolated. For example, peripheral blood mononuclearcells can be isolated from an immunized or naturally immune mammal,e.g., a human, and cultured for about 7 days in vitro. The PBMC culturesare then screened for specific IgGs that meet the screening criteria andthe cells from positive wells are isolated. Individual Ig-producing Bcells can be isolated by FACS or by identifying them in acomplement-mediated hemolytic plaque assay. Ig-producing B cells can bemicromanipulated into a tube and the V_(H) and V_(L) genes can beamplified using, e.g., RT-PCR. The V_(H) and V_(L) genes can be clonedinto an antibody expression vector and transfected into cells (e.g.,eukaryotic or prokaryotic cells) for expression.

Alternatively, antibody-producing cell lines can be selected andcultured using or routinely modifying techniques known in the art. Suchtechniques are described in a variety of laboratory manuals and primarypublications. In this respect, techniques suitable for use in theinvention as described below are described in Current Protocols inImmunology, Coligan et al., Eds., Green Publishing Associates andWiley-Interscience, John Wiley and Sons, New York (1991) which is hereinincorporated by reference in its entirety, including supplements.

Antibody fragments that recognize specific antigens and/or epitopes canbe generated using techniques known in the art. For example, Fab andF(ab′)₂ fragments can be produced recombinantly or by proteolyticcleavage of immunoglobulin molecules, using enzymes such as papain (toproduce Fab fragments) or pepsin (to produce F(ab′)₂ fragments). F(ab′)₂fragments contain the variable region, the light chain constant regionand the CH1 domain of the heavy chain. Such fragments are sufficient foruse, for example, in immunodiagnostic procedures involving coupling theimmunospecific portions of immunoglobulins to detecting reagents such asradioisotopes.

Completely human antibodies, such as those described herein, areparticularly desirable for therapeutic treatment of human patients.Human antibodies of the invention are isolated, e.g., from elderlyhealthy subjects who because of their age can be suspected to be at riskof developing a disorder, e.g., amyotrophic lateral sclerosis and/orfrontotemporal lobar degeneration, or a patient with the disorder butwith an unusually stable disease course. However, though it is prudentto expect that elderly healthy and symptom-free subjects, respectively,more regularly will have developed protective anti-TDP-43 antibodiesthan younger subjects, the latter can be used as well as a source forobtaining a human antibody of the invention. This is particularly truefor younger patients who are predisposed to develop a familial form of aTDP-43 proteinopathies but remain symptom-free since their immune systemand immune response functions more efficiently than that in olderadults.

Antibodies of the invention can be produced by any method known in theart for synthesis of antibodies, including in particular, chemicalsynthesis or recombinant expression techniques as described herein.

In one embodiment, an antibody, or antigen-binding fragment, variant, orderivative thereof of the invention comprises a synthetic constantregion wherein one or more domains are partially or entirely deleted(“domain-deleted antibodies”). In certain embodiments compatiblemodified antibodies will comprise domain deleted constructs or variantswherein the entire CH2 domain has been removed (ΔCH2 constructs). Forother embodiments a short connecting peptide can be substituted for thedeleted domain to provide flexibility and freedom of movement for thevariable region. Those of ordinary skill in the art will appreciate thatsuch constructs can be desirable due to the regulatory properties of theCH2 domain on the catabolic rate of the antibody. Domain deletedconstructs can be derived using a vector encoding an IgG₁ human constantdomain; see, e.g., International Applications WO02/060955 andWO02/096948A2. This vector is engineered to delete the CH2 domain andprovide a synthetic vector expressing a domain deleted IgG₁ constantregion.

In certain embodiments the antibodies (including antigen-bindingfragments, variants, or derivatives thereof) of the invention areminibodies. Minibodies can be made using methods known in the art; see,e.g., U.S. Pat. No. 5,837,821 or International Application PublicationNo. WO 94/09817.

In one embodiment, an antibody (e.g., antigen-binding fragment, variant,or derivative thereof of an antibody) of the invention comprises animmunoglobulin heavy chain having deletion or substitution of a few oreven a single amino acid as long as it permits association between themonomeric subunits. For example, the mutation of a single amino acid inselected areas of the CH2 domain can be enough to substantially reduceFc binding and thereby increase TDP-43 localization. Similarly, it canbe desirable to simply delete that part of one or more constant regiondomains that control the effector function (e.g., complement binding) tobe modulated. Such partial deletions of the constant regions can improveselected characteristics of the antibody (serum half-life) while leavingother desirable functions associated with the subject constant regiondomain intact. Moreover, as alluded to above, the constant regions ofthe disclosed antibodies can be synthetic through the mutation orsubstitution of one or more amino acids that enhances the profile of theresulting construct. In this respect it can be possible to disrupt theactivity provided by a conserved binding site (e.g. Fc binding) whilesubstantially maintaining the configuration and immunogenic profile ofthe modified antibody. Yet other embodiments comprise the addition ofone or more amino acids to the constant region to enhance desirablecharacteristics such as effector function or provide for more cytotoxinor carbohydrate attachment. In such embodiments it can be desirable toinsert or replicate specific sequences derived from selected constantregion domains.

The invention also provides antibodies that comprise, consistessentially of, or consist of, variants (including derivatives) ofantibodies (e.g., the V_(H) regions and/or V_(L) regions) describedherein, which antibodies (inducing antibody fragments),immunospecifically bind to TDP-43. Standard techniques known to those ofskill in the art can be used to introduce mutations in the nucleotidesequence encoding an antibody, including, but not limited to,site-directed mutagenesis and PCR-mediated mutagenesis which result inamino acid substitutions. In one embodiment, the variants (includingderivatives) encode less than 50 amino acid substitutions, less than 40amino acid substitutions, less than 30 amino acid substitutions, lessthan 25 amino acid substitutions, less than 20 amino acid substitutions,less than 15 amino acid substitutions, less than 10 amino acidsubstitutions, less than 5 amino acid substitutions, less than 4 aminoacid substitutions, less than 3 amino acid substitutions, or less than 2amino acid substitutions relative to the reference VH region,V_(H)-CDR1, V_(H)-CDR2, V_(H)-CDR3, VL region, V_(L)-CDR1, V_(L)-CDR2,or V_(L)-CDR3. A “conservative amino acid substitution” is one in whichthe amino acid residue is replaced with an amino acid residue having aside chain with a similar charge. Families of amino acid residues havingside chains with similar charges have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity (e.g., theability to bind TDP-43).

For example, it is possible to introduce mutations only in frameworkregions or only in CDR regions of an antibody. Introduced mutations canbe silent or neutral missense mutations, e.g., have no, or little,effect on an antibody's ability to bind antigen, indeed some suchmutations do not alter the amino acid sequence whatsoever. These typesof mutations can be useful to optimize codon usage, or improve ahybridoma's antibody production. Alternatively, non-neutral missensemutations can alter an antibody's ability to bind antigen. The locationof most silent and neutral missense mutations is likely to be in theframework regions, while the location of most non-neutral missensemutations is likely to be in a CDR, though this is not an absoluterequirement. A person of ordinary skill in the art is able to design andtest altered molecules for desired properties including for example,improvements in antigen-binding activity or change in antibodyspecificity. Following mutagenesis, proteins displaying the desiredproperties can routinely be expressed and the functional and/orbiological activity of the encoded protein, (e.g., ability toimmunospecifically bind at least one epitope of TDP-43) can bedetermined using or routinely modifying techniques known in the art.

Anti-TDP-43 antibodies of the present invention can be characterizedusing any in vivo or in vitro models of TDP-43 proteinopathies. Askilled artisan readily understands that an anti-TDP-43 antibody of theinvention can be characterized in a mouse model for TDP-43proteinopathies, for example, but not limited to, any one of the animalmodels for TDP-43 proteinopathies described in Example 5. Wegorzewska etal., Proc. Natl. Acad. Sci. U.S.A. 106 (2009), 18809-14; Gurney et al.,Science 264 (1994), 1772-75; Shan et al., Neuropharmacol. Letters 458(2009), 70-74; Wils et al., Proc. Natl. Acad. Sci. USA. 106 (2010),3858-63; Duchen and Strich, J. Neurol. Neurosurg. Psychiatry 31 (1968),535-42; Dennis and Citron, Neuroscience 185 (2009), 745-50; Swamp etal., Brain 134 (2011), 2610-2626; Igaz et al., J Clin Invest.121(2):726-38 (2011); Caccamo et al., Am J Pathol. 180(1):293-302(2012), Cannon et al., Acta Neuropathol. 123(6):807-23 (2012), Custer etal., Hum Mol Genet. 19(9):1741-55 (2010); and Tatom et al., Mol. Ther.17 (2009), 607-613.

A skilled artisan understands that an experimental model of TDP-43proteinopathy can be used in a preventative setting or it can be used ina therapeutic setting. In a preventative setting, the dosing of animalsstarts prior to the onset of the TDP-43 proteinopathy or symptomsthereof. In preventative settings, an anti-TDP-43 antibody of theinvention is evaluated for its ability to prevent, reduce or delay theonset of TDP-43 proteinopathy or symptoms thereof. In a therapeuticsetting, the dosing of animals start after the onset of TDP-43proteinopathy or a symptom thereof. In a therapeutic setting, ananti-TDP-43 antibody of the invention is evaluated for its ability totreat, reduce or alleviate the TDP-43 proteinopathy or a symptomthereof. Symptoms of the TDP-43 proteinopathies include, but are notlimited to, accumulation of pathological TDP-43 deposits, pathologicalTDP-43 distribution, phosphorylated TDP-43, or insoluble TDP-43fractions in the neurons, brain, spinal cord, cerebrospinal fluid orserum of the experimental object. A skilled artisan understands that apositive preventative or therapeutic outcome in any animal model ofTDP-43 proteinopathies indicates that the particular anti-TDP-43antibody can be used for preventative or therapeutic purposes in asubject other than the experimental model organism, for example, it canbe used to treat TDP-43 proteinopathies in a human subject in needthereof.

In one embodiment, an anti-TDP-43 antibody of the invention can beadministered to a TDP-43 proteinopathy mouse model and correspondingcontrol wild type mice. The antibody administered can be a murinizedantibody of the present invention or a human-murine chimera of anantibody of the present invention. The anti-TDP-43 antibody can beadministered by any means known in the art, for example, byintraperitoneal, intracranial, intramuscular, intravenous, subcutaneous,oral, and aerosol administration. Experimental animals can be given one,two, three, four, five or more doses of the anti-TDP-43 antibody or acontrol composition, such as PBS. In one embodiment, experimentalanimals will be administered one or two doses of an anti-TDP-43antibody. In another embodiment, the animals are chronically dosed withthe anti-TDP-43 antibody over several weeks or months. A skilled artisancan readily design a dosing regimen that fits the experimental purpose,for example, dosing regimen for acute studies, dosing regimen forchronic studies, dosing regimen for toxicity studies, dosing regimen forpreventative or therapeutic studies. The presence of the anti-TDP-43antibody in a particular tissue compartment of the experimental animals,for example, but not limited to, serum, blood, cerebrospinal fluid,brain tissue, can be established using well know methods of the art. Inone embodiment, an anti-TDP-43 antibody of the invention is capable topenetrate the blood brain barrier. In another embodiment, an anti-TDP-43antibody of the invention is capable to enter neurons. A skilled artisanunderstands that by adjusting the anti-TDP-43 antibody dose and thedosing frequency, a desired anti-TDP-43 antibody concentration can bemaintained in the experimental animals. Any effect of an anti-TDP-43antibody of the present invention in the TDP-43 proteinopathy models canbe assessed by comparing the level, biochemical characteristics ordistribution of TDP-43 in the treated and control animals. In oneembodiment, an antibody of the present invention is capable of reducingthe level, amount or concentration of TDP-43 inclusions in the brain orspinal cord in an animal model. The antibody can reduce the level,amount or concentration of TDP-43 inclusions by at least about 5%, 10%,20%, 30%, 50%, 70%, 90% or more. In another embodiment, an antibody ofthe present invention is capable of reducing the number or frequency ofTDP-43 inclusion-positive neurons in the brain or spinal cord in ananimal model, for example, by at least about 5%, 10%, 20%, 30%, 50%,70%, 90% or more. The effect of an antibody of the present invention canalso be assessed by examining the distribution and biochemicalproperties of TDP-43 following antibody administration. In oneembodiment, an antibody of the present invention is capable of reducingthe amount or concentration of cytoplasmic TDP-43 protein in the brainor spinal cord of an animal model, for example, by at least about 5%,10%, 20%, 30%, 50%, 70%, 90% or more. In another embodiment, an antibodyof the present invention is capable of reducing the amount orconcentration of neuritic TDP-43 protein in the brain or spinal cord ofan animal model, for example, by at least about 5%, 10%, 20%, 30%, 50%,70%, 90% or more. In a further embodiment, an antibody of the presentinvention can reduce the amount or concentration of phosphorylatedTDP-43 protein in the brain or spinal cord in an animal model, forexample, by at least about 5%, 10%, 20%, 30%, 50%, 70%, 90% or more.Phosphorylated TDP-43 can be detected using antibodies specific forpathologically phosphorylated forms of TDP-43, such as p403/p404 andp409/p410. Hasegawa et al., Ann Neurol, 64(1):60-70 (2008) An antibodyof the present invention can also alter, for example, reduce or increaseTDP-43 concentration in the blood, serum or cerebrospinal fluid of ananimal model, for example, by at least about 5%, 10%, 20%, 30%, 50%,70%, 90% or more. In one embodiment, the % reduction or increase isrelative compared to the level, number, frequency, amount orconcentration that existed before treatment, or to the level, number,frequency, amount or concentration that exist in an untreated/controltreated subject.

In one embodiment, an antibody of the present invention can prevent ordelay the onset of at least one symptom of a TDP-43 proteinopathy in asubject. In one embodiment, an antibody of the present invention canreduce or eliminate at least one symptom of a TDP-43 proteinopathy in asubject. The symptom can be the formation of pathological TDP-43deposits, phosphorylated TDP-43 deposits, or insoluble TDP-43 deposits.The symptom can also be the presence, or elevated concentration oramount, of TDP-43 in the serum, blood, urine or cerebrospinal fluid,wherein elevated concentration amount is compared to a healthy subject.The symptom can be a neurological symptom, for example, alteredconditioned taste aversion, altered contextual fear conditioning, memoryimpairment, loss of motor function. In one embodiment, memory impairmentis assessed using a two-trial Y-maze task. In one embodiment, the atleast one symptom is reduced by at least about 5%, 10%, 15%, 20%, 30%,50%, 70%, or 90%. In another embodiment, the two-trial Y-maze task ratiois significantly higher in an antibody treated subject than in a controlsubject. In a specific embodiment, the two-trial Y-maze task ratio isincreased by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,or 90%. In another embodiment, the two-trial Y-maze task ratio is atleast about two times, three times, four times, five times, ten times,or twenty times higher. The present invention also provides a method ofpreventing or delaying the onset of at least one symptom of a TDP-43proteinopathy in a subject in need thereof, comprising administering atherapeutically effective amount of an anti-TDP-43 antibody describedherein. The present invention further provides a method of reducing oreliminating least one symptom of a TDP-43 proteinopathy in a subject inneed thereof, comprising administering a therapeutically effectiveamount of an anti-TDP-43 antibody described herein. In one embodiment,the subject is an experimental organism, such as, but not limited to,transgenic mouse. In one embodiment, the subject is a human.

III. Polynucleotides Encoding Antibodies

A polynucleotide encoding an antibody, or antigen-binding fragment,variant, or derivative thereof can be composed of any polyribonucleotideor polydeoxribonucleotide, which can be unmodified RNA or DNA ormodified RNA or DNA. For example, a polynucleotide encoding an antibody,or antigen-binding fragment, variant, or derivative thereof can becomposed of single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that can be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, a polynucleotide encoding an antibody, orantigen-binding fragment, variant, or derivative thereof can be composedof triple-stranded regions comprising RNA or DNA or both RNA and DNA. Apolynucleotide encoding an antibody (including an antigen-bindingfragment of an antibody, or a variant, or derivative thereof) can alsocontain one or more modified bases or DNA or RNA backbones modified forstability or for other reasons. “Modified” bases include, for example,tritylated bases and unusual bases such as inosine. A variety ofmodifications can be made to DNA and RNA; thus, “polynucleotide”embraces chemically, enzymatically, or metabolically modified forms.

An isolated polynucleotide encoding a non-natural variant of apolypeptide derived from an immunoglobulin (e.g., an immunoglobulinheavy chain portion or light chain portion) can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence encoding the immunoglobulin such that oneor more amino acid substitutions, additions or deletions are introducedinto the encoded protein. Mutations can be introduced by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. In one embodiment, conservative amino acid substitutionsare made at one or more amino acid residue positions that arenon-essential.

RNA can be isolated from the original B cells, hybridoma cells or fromother transformed cells by standard techniques, such as guanidiniumisothiocyanate extraction and precipitation followed by centrifugationor chromatography. Where desirable, mRNA can be isolated from total RNAusing standard techniques such as, chromatography on oligo dT cellulose.Suitable techniques are familiar in the art. In one embodiment, cDNAsthat encode the light and the heavy chains of the antibody can be made,either simultaneously or separately, using reverse transcriptase and DNApolymerase in accordance with known methods. PCR can be initiated byconsensus constant region primers or by more specific primers based onthe published heavy and light chain DNA and amino acid sequences. Asdiscussed above, PCR also can be used to isolate DNA clones encoding theantibody light and heavy chains. In this case the libraries can bescreened by consensus primers or larger homologous probes, such as humanconstant region probes.

DNA, typically plasmid DNA, can be isolated from the cells usingtechniques known in the art, restriction mapped and sequenced inaccordance with standard, known techniques set forth in detail, e.g., inthe foregoing references relating to recombinant DNA techniques. Ofcourse, the DNA can be synthetic according to the invention at any pointduring the isolation process or subsequent analysis.

In one embodiment, the invention provides an isolated polynucleotidecomprising, consisting essentially of, or consisting of a nucleic acidencoding an immunoglobulin heavy chain variable region (V_(H)), where atleast one of the CDRs of the heavy chain variable region or at least twoof the V_(H)-CDRs of the heavy chain variable region are at least 80%,85%, 90%, 95%, 96%, 98%, or 99% identical to reference heavy chainV_(H)-CDR1, V_(H)-CDR2, or V_(H)-CDR3 amino acid sequences from theantibodies disclosed herein. Alternatively, the V_(H)-CDR1, V_(H)-CDR2,or V_(H)-CDR3 regions of the V_(H) are at least 80%, 85%, 90%, 95%, 96%,98%, or 99% identical to reference heavy chain V_(H)-CDR1, V_(H)-CDR2,and V_(H)-CDR3 amino acid sequences from the antibodies disclosedherein. Thus, according to this embodiment a heavy chain variable regionof the invention has V_(H)-CDR1, V_(H)-CDR2, or V_(H)-CDR3 polypeptidesequences related to the polypeptide sequences shown in FIGS. 1A-1K and3A-3R. In one embodiment, the amino acid sequence of the reference V_(H)CDR1 is SEQ ID NO: 3, 11, 19, 28, 37, 46, 54, 62, 70, 79, 88, 131, 139,147, 156, 164, 172, 180, 188, 196, 204, 212, 220, 228, 236, 244, 252,260, or 268; the amino acid sequence of the reference VH CDR2 is SEQ IDNO: 4, 12, 20, 29, 38, 47, 55, 63, 71, 80, 89, 132, 140, 148, 157, 165,173, 181, 189, 197, 205, 213, 221, 229, 237, 245, 253, 261, or 269; andthe amino acid sequence of the reference VH CDR3 is SEQ ID NO: 5, 13,21, 30, 39, 48, 56, 64, 72, 81, 90, 133, 141, 149, 158, 166, 174, 182,190, 198, 206, 214, 222, 230, 238, 246, 254, 262, or 270. In oneembodiment, the invention provides an isolated polynucleotidecomprising, consisting essentially of, or consisting of a nucleic acidencoding an immunoglobulin heavy chain variable region (VII), in whichthe V_(H)-CDR1, V_(H)-CDR2 and V_(H)-CDR3 regions have the polypeptidesequences of the V_(H)-CDR1, V_(H)-CDR2 and V_(H)-CDR3 groups shown inFIGS. 1A-1K and 3A-3R, except for one, two, three, four, five, six,seven, eight, nine, or ten amino acid substitutions in any oneV_(H)-CDR. In certain embodiments the amino acid substitutions areconservative. In one embodiment, the amino acid sequence of the VH CDR1is SEQ ID NO: 3, 11, 19, 28, 37, 46, 54, 62, 70, 79, 88, 131, 139, 147,156, 164, 172, 180, 188, 196, 204, 212, 220, 228, 236, 244, 252, 260, or268; the amino acid sequence of the VH CDR2 is SEQ ID NO: 4, 12, 20, 29,38, 47, 55, 63, 71, 80, 89, 132, 140, 148, 157, 165, 173, 181, 189, 197,205, 213, 221, 229, 237, 245, 253, 261, or 269; and the amino acidsequence of the VH CDR3 is SEQ ID NO: 5, 13, 21, 30, 39, 48, 56, 64, 72,81, 90, 133, 141, 149, 158, 166, 174, 182, 190, 198, 206, 214, 222, 230,238, 246, 254, 262, or 270.

In another embodiment, the invention provides an isolated polynucleotidecomprising, consisting essentially of, or consisting of a nucleic acidencoding an immunoglobulin light chain variable region (V_(L)), where atleast one of the V_(L)-CDRs of the light chain variable region or atleast two of the V_(L)-CDRs of the light chain variable region are atleast 80%, 85%, 90%, 95%, 96%, 98%, or 99% identical to reference lightchain V_(L)-CDR1, V_(L)-CDR2, or V_(L)-CDR3 amino acid sequences fromthe antibodies disclosed herein. Alternatively, the V_(L)-CDR1,V_(L)-CDR2, or V_(L)-CDR3 regions of the VL are at least 80%, 85%, 90%,95%, 96%, 98%, or 99% identical to reference light chain V_(L)-CDR1,V_(L)-CDR2, and V_(L)-CDR3 amino acid sequences from the antibodiesdisclosed herein. Thus, according to this embodiment a light chainvariable region of the invention has V_(L)-CDR1, V_(L)-CDR2, orV_(L)-CDR3 polypeptide sequences related to the polypeptide sequencesshown in FIGS. 1A-1K and 3A-3R. In one embodiment, the amino acidsequence of the reference VL CDR1 is SEQ ID NO: 7, 15, 23, 32, 42, 50,58, 66, 74, 84, 91, 135, 143, 152, 160, 168, 176, 184, 192, 200, 208,216, 224, 232, 240, 248, 256, 264, 272 or 326; the amino acid sequenceof the reference VL CDR2 is SEQ ID NO: 8, 16, 24, 33, 43, 51, 59, 67,75, 85, 92, 136, 144, 153, 161, 169, 177, 185, 193, 201, 209, 217, 225,233, 241, 249, 257, 265, 273 or 327; and the amino acid sequence of thereference VL CDR3 is SEQ ID NO: 9, 17, 25, 34, 44, 52, 60, 68, 76, 86,93, 137, 145, 154, 162, 170, 178, 186, 194, 202, 210, 218, 226, 234,242, 250, 258, 266, 274 or 328.

In another embodiment, the invention provides an isolated polynucleotidecomprising, consisting essentially of, or consisting of a nucleic acidencoding an immunoglobulin light chain variable region (V_(L)) in whichthe V_(L)-CDR1, V_(L)-CDR2 and V_(L)-CDR3 regions have the polypeptidesequences of the V_(L)-CDR1, V_(L)-CDR2 and V_(L)-CDR3 groups shown inFIGS. 1A-1K and 3A-3R, except for one, two, three, four, five, six,seven, eight, nine, or ten amino acid substitutions in any oneV_(L)-CDR. In certain embodiments the amino acid substitutions areconservative. In one embodiment, the amino acid sequence of the VL CDR1is SEQ ID NO: 7, 15, 23, 32, 42, 50, 58, 66, 74, 84, 91, 135, 143, 152,160, 168, 176, 184, 192, 200, 208, 216, 224, 232, 240, 248, 256, 264,272 or 326; the amino acid sequence of the VL CDR2 is SEQ ID NO: 8, 16,24, 33, 43, 51, 59, 67, 75, 85, 92, 136, 144, 153, 161, 169, 177, 185,193, 201, 209, 217, 225, 233, 241, 249, 257, 265, 273 or 327; and theamino acid sequence of the VL CDR3 is SEQ ID NO: 9, 17, 25, 34, 44, 52,60, 68, 76, 86, 93, 137, 145, 154, 162, 170, 178, 186, 194, 202, 210,218, 226, 234, 242, 250, 258, 266, 274 or 328.

As known in the art, “sequence identity” between two polypeptides or twopolynucleotides is determined by comparing the amino acid or nucleicacid sequence of one polypeptide or polynucleotide to the sequence of asecond polypeptide or polynucleotide. When discussed herein, whether anyparticular polypeptide is at least about 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90% or 95% identical to another polypeptide can bedetermined using methods and computer programs/software known in the artsuch as, but not limited to, the BESTFIT program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711).BESTFIT uses the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2 (1981), 482-489, to find the bestsegment of homology between two sequences. When using BESTFIT or anyother sequence alignment program to determine whether a particularsequence is, for example, 95% identical to a reference sequenceaccording to the invention, the parameters are set, of course, such thatthe percentage of identity is calculated over the full length of thereference polypeptide sequence and that gaps in homology of up to 5% ofthe total number of amino acids in the reference sequence are allowed.

In one embodiment of the invention, the polynucleotide comprises,consists essentially of, or consists of a nucleic acid having apolynucleotide sequence of the V_(H) or V_(L) region of an TDP-43binding antibody as listed in Table 3. In this respect, a person ofordinary skill in the art will readily appreciate that thepolynucleotides encoding at least the variable domain of the lightand/or heavy chain can encode the variable domain of both immunoglobulinchains or only one.

TABLE 3 Nucleotide sequences of the V_(H) and V_(L) region of TDP-43specific antibodies. Nucleotide sequences of variable heavy (VH)Antibody and variable light (VL) chains NI-205.3F10 V_(H) SEQ ID NO: 95V_(L) SEQ ID NO: 96 NI-205.51C1 V_(H) SEQ ID NO: 97 V_(L) SEQ ID NO: 98NI-205.21G2 V_(H) SEQ ID NO: 99 V_(L) SEQ ID NO: 100 NI-205.8A2 V_(H)SEQ ID NO: 101 V_(L) SEQ ID NO: 102 NI-205.15F12 V_(H) SEQ ID NO: 103V_(L) SEQ ID NO: 104 NI-205.113C4 V_(H) SEQ ID NO: 105 V_(L) SEQ ID NO:106 NI-205.25F3 V_(H) SEQ ID NO: 107 V_(L) SEQ ID NO: 108 NI-205.87E7V_(H) SEQ ID NO: 109 V_(L) SEQ ID NO: 110 NI-205.21G1 V_(H) SEQ ID NO:111 V_(L) SEQ ID NO: 112 NI-205.68G5 V_(H) SEQ ID NO: 113 V_(L) SEQ IDNO: 114 NI-205.20A1 V_(H) SEQ ID NO: 115 V_(L) SEQ ID NO: 116 NI205.41D1V_(H) SEQ. ID. NO: 275 V_(L) SEQ. ID. NO: 276 NI205.29E11 V_(H) SEQ. ID.NO: 277 V_(L) SEQ. ID. NO: 278 NI205.9E12 V_(H) SEQ. ID. NO: 279 V_(L)SEQ. ID. NO: 280 V_(L) SEQ. ID. NO: 281 NI205.98H6 V_(H) SEQ. ID. NO:282 V_(L) SEQ. ID. NO: 283 NI205.10D3 V_(H) SEQ. ID. NO: 284 V_(L) SEQ.ID. NO: 285 NI205.44B22 V_(H) SEQ. ID. NO: 286 V_(L) SEQ. ID. NO: 287NI205.38H2 V_(H) SEQ. ID. NO: 288 V_(L) SEQ. ID. NO: 289 NI205.36D5V_(H) SEQ. ID. NO: 290 V_(L) SEQ. ID. NO: 291 NI205.58E11 V_(H) SEQ. ID.NO: 292 V_(L) SEQ. ID. NO: 293 NI205.14H5 V_(H) SEQ. ID. NO: 294 V_(L)SEQ. ID. NO: 295 NI205.31D2 V_(H) SEQ. ID. NO: 296 V_(L) SEQ. ID. NO:297 NI205.8F8 V_(H) SEQ. ID. NO: 298 V_(L) SEQ. ID. NO: 299 NI205.31C11V_(H) SEQ. ID. NO: 300 V_(L) SEQ. ID. NO: 301 NI205.8C10 V_(H) SEQ. ID.NO: 302 V_(L) SEQ. ID. NO: 303 NI205.10H7 V_(H) SEQ. ID. NO: 304 V_(L)SEQ. ID. NO: 305 NI205.1A9 V_(H) SEQ. ID. NO: 306 V_(L) SEQ. ID. NO: 307NI205.14W3 V_(H) SEQ. ID. NO: 308 V_(L) SEQ. ID. NO: 309 NI205.19G5V_(H) SEQ. ID. NO: 310 V_(L) SEQ. ID. NO: 310

In one embodiment, the invention provides an isolated polynucleotidecomprising, consisting essentially of, or consisting of a nucleic acidencoding an immunoglobulin heavy chain variable region at least 80%,85%, 90%, 95%, 96%, 98%, or 99% or 95% identical to reference heavychain VH. In one embodiment, the amino acid sequence of the referenceheavy chain variable region is SEQ ID NO: 1, 10, 18, 26, 35, 45, 53, 61,69, 77, 87, 130, 138, 146, 155, 163, 171, 179, 187, 195, 203, 211, 219,227, 235, 243, 251, 259, or 267.

In one embodiment, the invention provides an isolated polynucleotidecomprising, consisting essentially of, or consisting of a nucleic acidencoding an immunoglobulin light chain variable region at least 80%,85%, 90%, 95%, 96%, 98%, or 99% or 95% identical to reference lightchain VL. In one embodiment, the amino acid sequence of the referencelight chain variable region is SEQ ID NO: 6, 14, 22, 31, 40, 49, 57, 65,73, 82, 122, 134, 142, 150, 151, 159, 167, 175, 183, 191, 199, 207, 215,223, 231, 239, 247, 255, 263, or 271.

The invention also includes fragments of the polynucleotides of theinvention. Preferably the polynucleotides encode a polypeptide thatbinds TDP-43. Additionally polynucleotides which encode fusionpolynucleotides, Fab fragments, and other derivatives, as describedherein, are also contemplated by the invention.

The polynucleotides can be produced or manufactured by any appropriatemethod known in the art. For example, if the nucleotide sequence of theantibody is known, a polynucleotide encoding the antibody can beassembled from chemically synthesized oligonucleotides, e.g., asdescribed in Kutmeier et al., BioTechniques 17 (1994), 242, which,briefly, involves the synthesis of overlapping oligonucleotidescontaining portions of the sequence encoding the antibody, annealing andligating of those oligonucleotides, and then amplification of theligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody (including anantigen-binding fragment of an antibody, or variant or derivativethereof) can be generated from nucleic acid from a suitable source. If aclone containing a nucleic acid encoding a particular antibody is notavailable, but the sequence of the antibody is known, a nucleic acidencoding the antibody can be chemically synthesized or obtained from asuitable source (e.g., an antibody cDNA library, or a cDNA librarygenerated from, or nucleic acid, preferably polyA⁺ RNA, isolated from,any tissue or cells expressing the TDP-43-specific antibody, such ashybridoma cells selected to express an antibody) by PCR amplificationusing synthetic primers hybridizable to the 3′ and 5′ ends of thesequence or by cloning using an oligonucleotide probe specific for theparticular gene sequence to identify, e.g., a cDNA clone from a cDNAlibrary that encodes the antibody. Amplified nucleic acids generated byPCR can then be cloned into replicable cloning vectors using any methodknown in the art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody, or antigen-binding fragment, variant, or derivativethereof is determined, its nucleotide sequence can be manipulated usingmethods known in the art for the manipulation of nucleotide sequences,e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc.(see, for example, the techniques described in Sambrook et al.,Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1990) and Ausubel et al., eds.,Current Protocols in Molecular Biology, John Wiley & Sons, NY (1998),the contents of both of which are herein incorporated by reference intheir entireties), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

IV. Expression of Antibody Polypeptides

Following manipulation of the isolated genetic material to provide anantibody (including an antigen-binding fragment of an antibody, orvariant or derivative thereof) of the invention, the polynucleotideencoding the antibody is typically inserted in an expression vector forintroduction into host cells that can be used to produce the desiredquantity of antibody. Recombinant expression of an antibody, orfragment, derivative or analog thereof, e.g., a heavy or light chain ofan antibody which binds to a target molecule is described herein. Once apolynucleotide encoding an antibody or a heavy or light chain of anantibody, or portion thereof (preferably containing the heavy or lightchain variable domain), of the invention has been obtained, the vectorfor the production of the antibody can be produced by recombinant DNAtechnology using techniques known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are known to those of ordinary skill in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody of the invention, or a heavy or light chainthereof, or a heavy or light chain variable domain, operably linked to apromoter. Such vectors can include the nucleotide sequence encoding theconstant region of the antibody (see, e.g., International ApplicationPublication No. WO 86/05807 and U.S. Pat. No. 5,122,464) and thevariable domain of the antibody can be cloned into such a vector forexpression of the entire heavy or light chain.

The term “vector” or “expression vector” is used herein to mean vectorsused in accordance with the invention as a vehicle for introducing intoand expressing a desired gene in a host cell. As known in the art, suchvectors can readily be selected from the group consisting of plasmids,phages, viruses and retroviruses. In general, vectors compatible withthe instant invention will comprise a selection marker, appropriaterestriction sites to facilitate cloning of the desired gene and theability to enter and/or replicate in eukaryotic or prokaryotic cells.Numerous expression vector systems can be employed for the purposes ofthis invention. For example, one class of vector utilizes DNA elementswhich are derived from animal viruses such as bovine papilloma virus,polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses(RSV, MMTV or MOMLV) or SV40 virus. Others involve the use ofpolycistronic systems with internal ribosome binding sites.Additionally, cells which have integrated the DNA into their chromosomescan be selected by introducing one or more markers which allow selectionof transfected host cells. The marker can provide for prototrophy to anauxotrophic host, biocide resistance (e.g., antibiotics) or resistanceto heavy metals such as copper. The selectable marker gene can either bedirectly linked to the DNA sequences to be expressed, or introduced intothe same cell by co-transformation. Additional elements can also beneeded for optimal synthesis of mRNA. These elements can include signalsequences, splice signals, as well as transcriptional promoters,enhancers, and termination signals.

In particular embodiments the cloned variable region genes are insertedinto an expression vector along with the heavy and light chain constantregion coding sequences (e.g., human heavy or light chain constantregion genes) as discussed above. In one embodiment, this is effectedusing a proprietary expression vector of Biogen IDEC, Inc., referred toas NEOSPLA, disclosed in U.S. Pat. No. 6,159,730. This vector containsthe cytomegalovirus promoter/enhancer, the mouse beta globin majorpromoter, the SV40 origin of replication, the bovine growth hormonepolyadenylation sequence, neomycin phosphotransferase exon 1 and exon 2,the dihydrofolate reductase gene and leader sequence. This vector hasbeen found to result in very high level expression of antibodies uponincorporation of variable and constant region genes, transfection in CHOcells, followed by selection in G418 containing medium and methotrexateamplification. Of course, any expression vector which is capable ofeliciting expression in eukaryotic cells can be used in the invention.Examples of suitable vectors include, but are not limited to plasmidspcDNA3, pHCMV/Zeo, pCR3.1, pEF1/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2,pTRACER-HCMV, pUB6N5-His, pVAX1, and pZeoSV2 (available from Invitrogen,San Diego, Calif.), and plasmid pCI (available from Promega, Madison,Wis.). In general, screening large numbers of transformed cells forthose which express suitably high levels if immunoglobulin heavy andlight chains is routine experimentation which can be carried out, forexample, by robotic systems. Vector systems are also taught in U.S. Pat.Nos. 5,736,137 and 5,658,570, each of which is herein incorporated byreference in its entirety. This system provides for high expressionlevels, e.g., >30 pg/cell/day. Other exemplary vector systems aredisclosed e.g., in U.S. Pat. No. 6,413,777.

In other embodiments the antibodies (e.g., antigen-binding fragments ofantibodies and variants or derivatives thereof) of the invention can beexpressed using polycistronic constructs such as those disclosed in U.S.Patent Application Publication No. 2003-0157641 A1 and hereinincorporated by reference in its entirety. In these expression systems,multiple gene products of interest such as heavy and light chains ofantibodies can be produced from a single polycistronic construct. Thesesystems advantageously use an internal ribosome entry site (IRES) toprovide relatively high levels of antibodies. Compatible IRES sequencesare disclosed in U.S. Pat. No. 6,193,980 which is also incorporatedherein. Those ordinary skill in the art will appreciate that suchexpression systems can be used to effectively produce the full range ofantibodies disclosed in the application.

More generally, once the vector or DNA sequence encoding a monomericsubunit of the antibody has been prepared, the expression vector can beintroduced into an appropriate host cell. Introduction of the plasmidinto the host cell can be accomplished by various techniques known tothose in the art. These include, but are not limited to, transfectionincluding lipotransfection using, e.g., Fugene or lipofectamine,protoplast fusion, calcium phosphate precipitation, cell fusion withenveloped DNA, microinjection, and infection with intact virus.Typically, plasmid introduction into the host is via standard methodsknown in the art, such as, calcium phosphate co-precipitation method.The host cells harboring the expression construct are grown underconditions appropriate to the production of the light chains and heavychains, and assayed for heavy and/or light chain protein synthesis.Non-limiting exemplary assay techniques include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), orfluorescence-activated cell sorter analysis (FACS), andimmunohistochemistry.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody for use in the methods describedherein. Thus, the invention includes host cells containing apolynucleotide encoding an antibody of the invention, or a heavy orlight chain thereof, operably linked to a heterologous promoter. Inparticular embodiments for the expression of double-chained antibodies,vectors encoding both the heavy and light chains can be co-expressed inthe host cell for expression of the entire immunoglobulin molecule, asdetailed below.

The host cell can be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors can contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector can be used which encodes both heavy and light chainpolypeptides. In such situations, the light chain is advantageouslyplaced before the heavy chain to avoid an excess of toxic free heavychain; see Proudfoot, Nature 322 (1986), 52; Kohler, Proc. Natl. Acad.Sci. USA 77 (1980), 2197. The coding sequences for the heavy and lightchains can comprise cDNA or genomic DNA.

As used herein, “host cells” refers to cells which harbor vectorsconstructed using recombinant DNA techniques and encoding at least oneheterologous gene. In descriptions of processes for isolation ofantibodies from recombinant hosts, the terms “cell” and “cell culture”are used interchangeably to denote the source of an antibody unless itis clearly specified otherwise. In other words, recovery of polypeptidefrom the “cells” can mean either from spun down whole cells, or from thecell culture containing both the medium and the suspended cells.

A variety of host-expression vector systems can be utilized to expressantibodies for use in methods described herein. Such host-expressionsystems represent vehicles by which the coding sequences of interest canbe produced and subsequently purified, but also represent cells whichcan, when transformed or transfected with the appropriate nucleotidecoding sequences, express an antibody of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, NSO, BLK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). In one embodiment, bacterial cells such as Escherichia coli,and eukaryotic cells, especially for the expression of whole recombinantantibody, are used for the expression of a recombinant antibody. Forexample, mammalian cells such as Chinese Hamster Ovary (CHO) cells, inconjunction with a vector such as the major intermediate early genepromoter element from human cytomegalovirus is an effective expressionsystem for antibodies; see, e.g., Foecking et al., Gene 45 (1986), 101;Cockett et al., Bio/Technology 8 (1990), 2.

The host cell lines used for protein expression of the TDP-43 bindingmolecules of the invention include, for example cells of mammalianorigin; those of ordinary skill in the art are credited with ability todetermine particular host cell lines which are best suited for thedesired gene product to be expressed therein. Exemplary host cell linesinclude, but are not limited to, CHO (Chinese Hamster Ovary), DG44 andDUXB11 (Chinese Hamster Ovary lines, DHFR minus), HELA (human cervicalcarcinoma), CVI (monkey kidney line), COS (a derivative of CVI with SV40T antigen), VERY, BHK (baby hamster kidney), MDCK, WI38, R1610 (Chinesehamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidneyline), SP2/O (mouse myeloma), P3×63-Ag3.653 (mouse myeloma), BFA-1c1BPT(bovine endothelial cells), RAJI (human lymphocyte) and 293 (humankidney). In a specific embodiment, host cell lines are CHO or 293 cells.Host cell lines are readily available and are typically available fromcommercial services, including for example, the American Tissue CultureCollection or from published literature.

In addition, a host cell strain can be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products canbe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product can be used.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody can be engineered. Rather than using expression vectorswhich contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells can beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method canadvantageously be used to engineer cell lines which stably express theantibody.

A number of selection systems can be used according to the presentinvention, including but not limited to, the herpes simplex virusthymidine kinase (Wigler et al., Cell 11 (1977), 223),hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski,Proc. Natl. Acad. Sci. USA 48 (1992), 202), and adeninephosphoribosyltransferase (Lowy et al., Cell 22 (1980), 817) genes canbe employed in tk-, hgprt- or aprt-cells, respectively. Also,anti-metabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77 (1980), 357; O'Hare et al., Proc. Natl.Acad. Sci. USA 78 (1981), 1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78(1981), 2072); neo, which confers resistance to the aminoglycoside G-418Goldspiel et al., Clinical Pharmacy 12 (1993), 488-505; Wu and Wu,Biotherapy 3 (1991), 87-95; Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32(1993), 573-596; Mulligan, Science 260 (1993), 926-932; and Morgan andAnderson, Ann. Rev. Biochem. 62 (1993), 191-217; TIB TECH 11 (1993),155-215; and hygro, which confers resistance to hygromycin (Santerre etal., Gene 30 (1984), 147. Methods known in the art of recombinant DNAtechnology which can be used are described in Ausubel et al., (eds.),Current Protocols in Molecular Biology, John Wiley & Sons, N Y (1993);Kriegler, Gene Transfer and Expression, A Laboratory Manual, StocktonPress, N Y (1990); and in Chapters 12 and 13, Dracopoli et al., (eds),Current Protocols in Human Genetics, John Wiley & Sons, N Y (1994);Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), each of which areherein incorporated by reference in their entireties.

The expression levels of an antibody can be increased using techniquesknown in the art, including for example, by vector amplification, seee.g., Bebbington and Hentschel, The use of vectors based on geneamplification for the expression of cloned genes in mammalian cells inDNA cloning, Academic Press, New York, Vol. 3. (1987). When a marker inthe vector system expressing an antibody is amplifiable, increase in thelevel of inhibitor present in culture of host cell will increase thenumber of copies of the marker gene. Since the amplified region isassociated with the antibody gene, production of the antibody will alsoincrease; see Crouse et al., Mol. Cell. Biol. 3 (1983), 257.

In vitro production allows scale-up to give large amounts of the desiredpolypeptides. Techniques for mammalian cell cultivation under tissueculture conditions are known in the art and include homogeneoussuspension culture, e.g. in an airlift reactor or in a continuousstirrer reactor, or immobilized or entrapped cell culture, e.g. inhollow fibers, microcapsules, on agarose microbeads or ceramiccartridges. If necessary and/or desired, the solutions of polypeptidescan be purified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose or (immuno-)affinity chromatography, e.g., afterpreferential biosynthesis of a synthetic hinge region polypeptide orprior to or subsequent to the HIC chromatography step described herein.

Genes encoding antibodies (including for example, antigen-bindingfragments of antibodies and variants, or derivatives thereof) of theinvention can also be expressed in non-mammalian cells such as bacteriaor insect or yeast or plant cells. Bacteria which readily take upnucleic acids include members of the enterobacteriaceae, such as strainsof Escherichia coli or Salmonella; Bacillaceae, such as Bacillussubtilis; Pneumococcus; Streptococcus, and Haemophilus influenzae. Itwill further be appreciated that, when expressed in bacteria, theheterologous polypeptides typically become part of inclusion bodies. Theheterologous polypeptides must be isolated, purified and then assembledinto functional molecules. Where tetravalent forms of antibodies aredesired, the subunits will then self-assemble into tetravalentantibodies; see, e.g., International Application Publication No.WO02/096948.

In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for the antibodybeing expressed. For example, when a large quantity of such a protein isto be produced, for the generation of pharmaceutical compositions of anantibody, vectors which direct the expression of high levels of fusionprotein products that are readily purified can be desirable. Suchvectors include, but are not limited, to the E. coli expression vectorpUR278 (Ruther et al., EMBO J. 2 (1983), 1791), in which the antibodycoding sequence can be ligated individually into the vector in framewith the lacZ coding region so that a fusion protein is produced; pINvectors (Inouye & Inouye, Nucleic Acids Res. 13 (1985), 3101-3109; VanHeeke & Schuster, J. Biol. Chem. 24 (1989), 5503-5509); and the like.pGEX vectors can also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption and binding to a matrix of glutathione-agarose beads followedby elution in the presence of free glutathione. The pGEX vectors aredesigned to include thrombin or factor Xa protease cleavage sites sothat the cloned target gene product can be released from the GST moiety.

In addition to prokaryotes, eukaryotic microbes can also be used.Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among eukaryotic microorganisms although a number of other strainsare commonly available, e.g., Pichia pastoris. For expression inSaccharomyces, the plasmid YRp7, for example, (Stinchcomb et al., Nature282 (1979), 39; Kingsman et al., Gene 7 (1979), 141; Tschemper et al.,Gene 10 (1980), 157) is commonly used. This plasmid already contains theTRP 1 gene which provides a selection marker for a mutant strain ofyeast lacking the ability to grow in tryptophan, for example ATCC No.44076 or PEP4-1 (Jones, Genetics 85 (1977), 12). The presence of thetrpl lesion as a characteristic of the yeast host cell genome thenprovides an effective environment for detecting transformation by growthin the absence of tryptophan.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is typically used as a vector to express foreign genes. Thevirus grows in Spodoptera frugiperda cells. The antibody coding sequencecan be cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

Once an antibody of the invention has been recombinantly expressed, thewhole antibodies, their dimers, individual light and heavy chains, orother immunoglobulin forms of the invention, can be purified accordingto standard procedures of the art, including for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, e.g. ammoniumsulfate precipitation, or by any other standard technique for thepurification of proteins; see, e.g., Scopes, “Protein Purification”,Springer Verlag, N.Y. (1982). Alternatively, another method forincreasing the affinity of antibodies of the invention is disclosed inU.S. Patent Publication No. 2002-0123057 A1.

V. Fusion Proteins and Conjugates

In certain embodiments, the antibody polypeptide comprises an amino acidsequence or one or more moieties not normally associated with anantibody. Exemplary modifications are described in more detail below.For example, a single-chain AT antibody fragment of the invention cancomprise a flexible linker sequence, or can be modified to add afunctional moiety (e.g., PEG, a drug, a toxin, or a label such as afluorescent, radioactive, enzyme, nuclear magnetic, heavy metal and thelike)

An antibody polypeptide of the invention can comprise, consistessentially of, or consist of a fusion protein. Fusion proteins arechimeric molecules which comprise, for example, an immunoglobulinTDP-43-binding domain with at least one target binding site, and atleast one heterologous portion, i.e., a portion with which it is notnaturally linked in nature. The amino acid sequences can normally existin separate proteins that are brought together in the fusion polypeptideor they can normally exist in the same protein but are placed in a newarrangement in the fusion polypeptide. Fusion proteins can be created,for example, by chemical synthesis, or by creating and translating apolynucleotide in which the peptide regions are encoded in the desiredrelationship.

The term “heterologous” as applied to a polynucleotide or a polypeptide,means that the polynucleotide or polypeptide is derived from a distinctentity from that of the rest of the entity to which it is beingcompared. For instance, as used herein, a “heterologous polypeptide” tobe fused to an antibody, or an antigen-binding fragment, variant, oranalog thereof is derived from a non-immunoglobulin polypeptide of thesame species, or an immunoglobulin or non-immunoglobulin polypeptide ofa different species.

As discussed in more detail elsewhere herein, antibodies, (e.g.,antigen-binding fragments of antibodies and variants or derivativesthereof) or antigen-binding fragments, variants, or derivatives thereofof the invention can further be recombinantly fused to a heterologouspolypeptide at the N- or C-terminus or chemically conjugated (includingcovalent and non-covalent conjugations) to polypeptides or othercompositions. For example, antibodies can be recombinantly fused orconjugated to molecules useful as labels in detection assays andeffector molecules such as heterologous polypeptides, drugs,radionuclides, or toxins; see, e.g., International ApplicationPublication Nos. WO92/08495; WO91/14438; WO89/12624; U.S. Pat. No.5,314,995; and European Patent Application No. EP 0 396 387.

Antibodies (e.g., antigen-binding fragments of antibodies and variants,or derivatives thereof) of the invention can be composed of amino acidsjoined to each other by peptide bonds or modified peptide bonds, i.e.,peptide isosteres, and can contain amino acids other than the 20gene-encoded amino acids. Antibodies can be modified by naturalprocesses, such as posttranslational processing, or by chemicalmodification techniques which are known in the art. Such modificationsare well described in basic texts and in more detailed monographs, aswell as in a voluminous research literature. Modifications can occuranywhere in the antibody, including the peptide backbone, the amino acidside-chains and the amino or carboxyl termini, or on moieties such ascarbohydrates. It will be appreciated that the same type of modificationcan be present in the same or varying degrees at several sites in agiven antibody. Also, a given antibody can contain many types ofmodifications. Antibodies can be branched, for example, as a result ofubiquitination, and they can be cyclic, with or without branching.Cyclic, branched, and branched cyclic antibodies can result fromposttranslation natural processes or can be made by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination; see, e.g.,Proteins—Structure And Molecular Properties, T. E. Creighton, W. H.Freeman and Company, New York 2nd Ed., (1993); PosttranslationalCovalent Modification Of Proteins, B. C. Johnson, Ed., Academic Press,New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol. 182 (1990),626-646; Rattan et al., Ann. NY Acad. Sci. 663 (1992), 48-62).

The invention also provides for fusion proteins comprising an antibody,or antigen-binding fragment, variant, or derivative thereof, and aheterologous polypeptide. In one embodiment, a fusion protein of theinvention comprises, consists essentially of, or consists of, apolypeptide having the amino acid sequence of any one or more of theV_(H) regions of an antibody of the invention or the amino acid sequenceof any one or more of the V_(L) regions of an antibody of the inventionor fragments or variants thereof, and a heterologous polypeptidesequence. In another embodiment, a fusion protein for use in thediagnostic and treatment methods disclosed herein comprises, consistsessentially of, or consists of a polypeptide having the amino acidsequence of any one, two, three of the V_(H)-CDRs of an antibody, orfragments, variants, or derivatives thereof, or the amino acid sequenceof any one, two, three of the V_(L)-CDRs of an antibody, or fragments,variants, or derivatives thereof, and a heterologous polypeptidesequence. In one embodiment, the fusion protein comprises a polypeptidehaving the amino acid sequence of a V_(H)-CDR3 of an antibody of theinvention, or fragment, derivative, or variant thereof, and aheterologous polypeptide sequence, which fusion protein specificallybinds to TDP-43. In another embodiment, a fusion protein comprises apolypeptide having the amino acid sequence of at least one V_(H) regionof an antibody of the invention and the amino acid sequence of at leastone VL region of an antibody of the invention or fragments, derivativesor variants thereof, and a heterologous polypeptide sequence. In oneembodiment, the V_(H) and V_(L) regions of the fusion protein correspondto a single source antibody (or scFv or Fab fragment) which specificallybinds TDP-43. In yet another embodiment, a fusion protein for use in thediagnostic and treatment methods disclosed herein comprises apolypeptide having the amino acid sequence of any one, two, three ormore of the V_(H) CDRs of an antibody and the amino acid sequence of anyone, two, three or more of the V_(L) CDRs of an antibody, or fragmentsor variants thereof, and a heterologous polypeptide sequence. In oneembodiment, two, three, four, five, six, or more of the V_(H)-CDR(s) orV_(L)-CDR(s) correspond to single source antibody (or scFv or Fabfragment) of the invention. Nucleic acid molecules encoding these fusionproteins are also encompassed by the invention.

Exemplary fusion proteins reported in the literature include fusions ofthe T cell receptor (Gascoigne et al., Proc. Natl. Acad. Sci. USA 84(1987), 2936-2940; CD4 (Capon et al., Nature 337 (1989), 525-531;Traunecker et al., Nature 339 (1989), 68-70; Zettmeissl et al., DNACell. Biol. USA 9 (1990), 347-353; and Byrn et al., Nature 344 (1990),667-670); L-selectin (homing receptor) (Watson et al., J. Cell. Biol.110 (1990), 2221-2229; and Watson et al., Nature 349 (1991), 164-167);CD44 (Aruffo et al., Cell 61 (1990), 1303-1313); CD28 and B7 (Linsley etal., J. Exp. Med. 173 (1991), 721-730); CTLA-4 (Lisley et al., J. Exp.Med. 174 (1991), 561-569); CD22 (Stamenkovic et al., Cell 66 (1991),1133-1144); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA88 (1991), 10535-10539; Lesslauer et al., Eur. J. Immunol. 27 (1991),2883-2886; and Peppel et al., J. Exp. Med. 174 (1991), 1483-1489 (1991);and IgE receptor a (Ridgway and Gorman, J. Cell. Biol. 115 (1991),Abstract No. 1448).

As discussed elsewhere herein, antibodies, (e.g., intact antibodies, andantigen-binding fragments of antibodies and variants, or derivativesthereof) of the invention can be fused to heterologous polypeptides toincrease the in vivo half-life of the polypeptides or for use inimmunoassays using methods known in the art. For example, in oneembodiment, PEG can be conjugated to the antibodies of the invention toincrease their half-life in vivo; see, e.g., Leong et al., Cytokine 16(2001), 106-119; Adv. in Drug Deliv. Rev. 54 (2002), 531; or Weir etal., Biochem. Soc. Transactions 30 (2002), 512.

Moreover, antibodies (e.g., intact antibodies, and antigen-bindingfragments of antibodies and variants, or derivatives thereof) of theinvention can be fused to marker sequences, such as a peptide tofacilitate their purification or detection. In particular embodiments,the marker amino acid sequence is a hexa-histidine peptide (HIS), suchas the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue,Chatsworth, Calif., 91311), among others, many of which are commerciallyavailable. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86(1989), 821-824, for instance, hexa-histidine provides for convenientpurification of the fusion protein. Other peptide tags useful forpurification include, but are not limited to, the “HA” tag, whichcorresponds to an epitope derived from the influenza hemagglutininprotein (Wilson et al., Cell 37 (1984), 767) and the “flag” tag.

Fusion proteins can be prepared using methods that are well known in theart; see for example U.S. Pat. Nos. 5,116,964 and 5,225,538. The precisesite at which the fusion is made can be selected empirically to optimizethe secretion or binding characteristics of the fusion protein. DNAencoding the fusion protein is then transfected into a host cell forexpression.

Antibodies of the invention can be used in non-conjugated form or can beconjugated to at least one of a variety of molecules, e.g., to improvethe therapeutic properties of the molecule, to facilitate targetdetection, or for imaging or therapy of the patient. Antibodies (e.g.,intact antibodies, and antigen-binding fragments of antibodies andvariants, or derivatives thereof) of the invention can be labeled orconjugated either before or after purification, when purification isperformed. In particular, antibodies of the invention can be conjugatedto therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses,lipids, biological response modifiers, pharmaceutical agents, or PEG.

Conjugates that are immunotoxins including conventional antibodies havebeen widely described in the art. The toxins can be coupled to theantibodies by conventional coupling techniques or immunotoxinscontaining protein toxin portions can be produced as fusion proteins.The antibodies of the invention can be used in a corresponding way toobtain such immunotoxins. Illustrative of such immunotoxins are thosedescribed by Byers, Seminars Cell. Biol. 2 (1991), 59-70 and by Fanger,Immunol. Today 12 (1991), 51-54.

Those of ordinary skill in the art will appreciate that conjugates canalso be assembled using a variety of techniques depending on theselected agent to be conjugated. For example, conjugates with biotin areprepared e.g. by reacting an TDP-43 binding polypeptide with anactivated ester of biotin such as the biotin N-hydroxysuccinimide ester.Similarly, conjugates with a fluorescent marker can be prepared in thepresence of a coupling agent, e.g. those listed herein, or by reactionwith an isothiocyanate, or fluorescein-isothiocyanate. Conjugates of theof the invention are prepared in an analogous manner.

The invention further encompasses antibodies (e.g., intact antibodies,and antigen-binding fragments of antibodies and variants, or derivativesthereof) of the invention conjugated to a diagnostic or therapeuticagent. The antibodies can be used diagnostically to, for example,demonstrate presence of a neurological disease, to indicate the risk ofgetting a neurological disease, to monitor the development orprogression of a neurological disease, i.e. TDP-43 proteinopathy as partof a clinical testing procedure to, e.g., determine the efficacy of agiven treatment and/or prevention regimen. Detection can be facilitatedby coupling the antibody, or antigen-binding fragment, variant, orderivative thereof to a detectable substance. Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, radioactivematerials, positron emitting metals using various positron emissiontomographies, and nonradioactive paramagnetic metal ions; see, e.g.,U.S. Pat. No. 4,741,900 for metal ions which can be conjugated toantibodies for use as diagnostics according to the invention. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ¹¹¹Inor ⁹⁹Tc.

An antibody, or antigen-binding fragment, variant, or derivative thereofalso can be detectably labeled by coupling it to a chemiluminescentcompound. The presence of the chemiluminescent-tagged antibody is thendetermined by detecting the presence of luminescence that arises duringthe course of a chemical reaction. Examples of particularly usefulchemiluminescent labeling compounds are luminol, isoluminol, theromaticacridinium ester, imidazole, acridinium salt and oxalate ester.

One of the ways in which an antibody, or antigen-binding fragment,variant, or derivative thereof can be detectably labeled is by linkingthe same to an enzyme and using the linked product in an enzymeimmunoassay (EIA) (Voller, A., “The Enzyme Linked Immunosorbent Assay(ELISA)” Microbiological Associates Quarterly Publication, Walkersville,Md., Diagnostic Horizons 2 (1978), 1-7); Voller et al., J. Clin. Pathol.31 (1978), 507-520; Butler, Meth. Enzymol. 73 (1981), 482-523; Maggio,E. (ed.), Enzyme Immunoassay, CRC Press, Boca Raton, Fla., (1980);Ishikawa, E. et al., (eds.), Enzyme Immunoassay, Kgaku Shoin, Tokyo(1981). The enzyme, which is bound to the antibody will react with anappropriate substrate, preferably a chromogenic substrate, in such amanner as to produce a chemical moiety which can be detected, forexample, by spectrophotometric, fluorimetric or by visual means. Enzymeswhich can be used to detectably label the antibody include, but are notlimited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. Additionally, the detection can be accomplished bycolorimetric methods which employ a chromogenic substrate for theenzyme. Detection can also be accomplished by visual comparison of theextent of enzymatic reaction of a substrate in comparison with similarlyprepared standards.

Detection can also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibody, orantigen-binding fragment, variant, or derivative thereof, it is possibleto detect the antibody through the use of a radioimmunoassay (RIA) (see,for example, Weintraub, B., Principles of Radioimmunoassays, SeventhTraining Course on Radioligand Assay Techniques, The Endocrine Society,(March, 1986)), which is herein incorporated by reference in itsentirety). The radioactive isotope can be detected by means including,but not limited to, a gamma counter, a scintillation counter, orautoradiography.

An antibody, or antigen-binding fragment, variant, or derivative thereofcan also be detectably labeled using fluorescence emitting metals suchas ¹⁵²Eu, or others of the lanthanide series. These metals can beattached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

Techniques for conjugating various moieties to an antibody, orantigen-binding fragment, variant, or derivative thereof are known; see,e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of DrugsIn Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy,Reisfeld et al., (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985);Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled DrugDelivery (2nd Ed.), Robinson et al., (eds.), Marcel Dekker, Inc., pp.623-53 (1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In CancerTherapy: A Review”, in Monoclonal Antibodies '84: Biological AndClinical Applications, Pinchera et al., (eds.), pp. 475-506 (1985);“Analysis, Results, And Future Prospective Of The Therapeutic Use OfRadiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies ForCancer Detection And Therapy, Baldwin et al., (eds.), Academic Press pp.303-16 (1985), and Thorpe et al., “The Preparation And CytotoxicProperties Of Antibody-Toxin Conjugates”, Immunol. Rev. 62 (1982),119-158.

As mentioned, in certain embodiments, a moiety that enhances thestability or efficacy of a binding molecule, e.g., a bindingpolypeptide, e.g., an antibody or immunospecific fragment thereof can beconjugated. For example, in one embodiment, PEG can be conjugated to thebinding molecules of the invention to increase their half-life in vivo.Leong et al., Cytokine 16 (2001), 106; Adv. in Drug Deliv. Rev. 54(2002), 531; or Weir et al., Biochem. Soc. Transactions 30 (2002), 512.

VI. Compositions and Methods of Use

The invention relates to compositions comprising the aforementionedTDP-43 binding molecule, e.g., antibody or antigen-binding fragmentthereof of the invention or derivative or variant thereof, or thepolynucleotide, vector or cell of the invention. The composition of theinvention can further comprise a pharmaceutically acceptable carrier.Furthermore, the pharmaceutical composition of the invention cancomprise further agents such as interleukins or interferons depending onthe intended use of the pharmaceutical composition. For example, for usein the treatment of TDP-43 proteinopathy the additional agent can beselected from the group consisting of small organic molecules,anti-TDP-43 antibodies, and combinations thereof. Hence, in a particularembodiment the invention relates to the use of the TDP-43 bindingmolecule, e.g., antibody or antigen-binding fragment thereof of theinvention or of a binding molecule having substantially the same bindingspecificities of any one thereof, the polynucleotide, the vector or thecell of the invention for the preparation of a pharmaceutical ordiagnostic composition for prophylactic and therapeutic treatment of aTDP-43 proteinopathy, monitoring the progression of a TDP-43proteinopathy or a response to a TDP-43 proteinopathy treatment in asubject or for determining a subject's risk for developing a TDP-43proteinopathy.

Hence, in one embodiment the invention relates to a method of treating aneurological disorder characterized by abnormal accumulation and/ordeposition of TDP-43 in the brain and the central nervous system,respectively, which method comprises administering to a subject in needthereof a therapeutically effective amount of any one of theafore-described TDP-43 binding molecules, antibodies, polynucleotides,vectors or cells of the instant invention. The term “TDP-43proteinopathy” includes but is not limited to TDP-43 proteinopathiessuch as argyrophilic grain disease, Alzheimer's disease, Amyotrophiclateral sclerosis (ALS), ALS-Parkinsonism dementia complex of Guam,corticobasal degeneration, Dementia with Lewy bodies, Huntington'sdisease, Lewy body disease, motor neuron disease, frontotemporal lobardegeneration (FTLD), frontotemporal dementia, frontotemporal lobardegeneration with ubiquitin-positive inclusions, hippocampal sclerosis,inclusion body myopathy, inclusion body myositis, Parkinson's disease,Parkinson's disease dementia, Parkinson-dementia complex in Kiipeninsula and Pick's disease as well as other movement disorders,neurodegenerative diseases and disease of the central nervous system(CNS) in general. Unless stated otherwise, the terms neurodegenerative,neurological or neuropsychiatric are used interchangeably herein.

A particular advantage of the therapeutic approach of the invention liesin the fact that the antibodies of the invention are derived from Bcells or B memory cells from healthy human subjects with no signs of ALSand/or FTLD and thus are, with a certain probability, capable ofpreventing a clinically manifest TDP-43 proteinopathies, or ofdiminishing the risk of the occurrence of the clinically manifestdisease, or of delaying the onset of the clinically manifest disease.Typically, the antibodies of the invention also have alreadysuccessfully gone through somatic maturation, i.e. the optimization withrespect to target selectivity and effectiveness in the high affinitybinding to the target TDP-43 molecule by means of somatic variation ofthe variable regions of the antibody.

The knowledge that such cells in vivo, e.g., in a human, have not beenactivated by means of related or other physiological proteins or cellstructures in the sense of an autoimmunological or allergic reaction isalso of great medical importance since this signifies a considerablyincreased chance of successfully living through the clinical testphases. So to speak, efficiency, acceptability and tolerability havealready been demonstrated before the preclinical and clinicaldevelopment of the prophylactic or therapeutic antibody in at least onehuman subject. It can thus be expected that the human anti-TDP-43antibodies of the invention, both its target structure-specificefficiency as therapeutic agent and its decreased probability of sideeffects significantly increase its clinical probability of success.

The invention also provides a pharmaceutical and diagnostic,respectively, pack or kit comprising one or more containers filled withone or more of the above described ingredients, e.g. anti-TDP-43antibody, binding fragment, derivative or variant thereof,polynucleotide, vector or cell of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition oralternatively the kit comprises reagents and/or instructions for use inappropriate diagnostic assays. The composition, e.g. kit of theinvention is of course particularly suitable for the risk assessment,diagnosis, prevention and treatment of a disorder which is accompaniedwith the presence of TDP-43, and in particular applicable for thetreatment of neurodegenerative diseases, TDP-43 proteinopathies,argyrophilic grain disease, Alzheimer's disease, Amyotrophic lateralsclerosis (ALS), ALS-Parkinsonism dementia complex of Guam, corticobasaldegeneration, Dementia with Lewy bodies, Huntington's disease, Lewy bodydisease, motor neuron disease, frontotemporal lobar degeneration (FTLD),frontotemporal dementia, frontotemporal lobar degeneration withubiquitin-positive inclusions, hippocampal sclerosis, inclusion bodymyopathy, inclusion body myositis, Parkinson's disease, Parkinson'sdisease dementia, Parkinson-dementia complex in Kii peninsula and Pick'sdisease.

In a specific embodiment, the composition of invention is in a sterileaqueous solution. In another embodiment, one or more of the componentsof a composition of the invention have been lyophilized. In anadditional embodiment, the composition of the invention does not containserum. In a further embodiment, one or more antibodies contained in acomposition of the invention are recombinantly produced. In anotherembodiment, the population of antibodies of the invention in thecomposition constitutes at least 10% of the immunoglobulin population inthe composition.

The pharmaceutical compositions of the invention can be formulatedaccording to methods known in the art; see for example Remington: TheScience and Practice of Pharmacy (2000) by the University of Sciences inPhiladelphia, ISBN 0-683-306472. Examples of suitable pharmaceuticalcarriers are known in the art and include phosphate buffered salinesolutions, water, emulsions, such as oil/water emulsions, various typesof wetting agents, sterile solutions etc. Compositions comprising suchcarriers can be formulated by known conventional methods. Thesepharmaceutical compositions can be administered to the subject at asuitable dose. Administration of the suitable compositions can beeffected by different ways, e.g., by intravenous, intraperitoneal,subcutaneous, intramuscular, topical or intradermal administration.Aerosol formulations such as nasal spray formulations include purifiedaqueous or other solutions of the active agent with preservative agentsand isotonic agents. Such formulations are adjusted to a pH and isotonicstate compatible with the nasal mucous membranes. Formulations forrectal or vaginal ad-ministration can be presented as a suppository witha suitable carrier.

More particularly, pharmaceutical compositions suitable for injectableuse include sterile aqueous solutions (where water soluble) ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions. In such cases, thecomposition must be sterile and should be fluid to the extent that easysyringability exists. It should be stable under the conditions ofmanufacture and storage and will preferably be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants.

Furthermore, whereas the invention includes the now standard (thoughfortunately infrequent) procedure of drilling a small hole in the skullto administer a drug of the invention, in one aspect, the bindingmolecule, especially antibody or antibody based drug of the inventioncan cross the blood-brain barrier, which allows for intravenous or oraladministration.

The dosage regimen will be determined by the attending physician andclinical factors. As is known in the medical arts, dosages for any onepatient depends upon many factors, including the patient's size, bodysurface area, age, the particular compound to be administered, sex, timeand route of administration, general health, and other drugs beingadministered concurrently. A typical dose can be, for example, in therange of 0.001 to 1000 μg (or of nucleic acid for expression or forinhibition of expression in this range); however, doses below or abovethis exemplary range are envisioned, especially considering theaforementioned factors. Generally, the dosage can range, e.g., fromabout 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg (e.g., 0.02mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.), ofthe host body weight. For example dosages can be 1 mg/kg body weight or10 mg/kg body weight or within the range of 1-10 mg/kg, or at least 1mg/kg. Doses intermediate in the above ranges are also intended to bewithin the scope of the invention. Subjects can be administered suchdoses daily, on alternative days, weekly or according to any otherschedule determined by empirical analysis. An exemplary treatmententails administration in multiple dosages over a prolonged period, forexample, of at least six months. Additional exemplary treatment regimensentail administration once per every two weeks or once a month or onceevery 3 to 6 months. Exemplary dosage schedules include 1-10 mg/kg or 15mg/kg on consecutive days, 30 mg/kg on alternate days or 60 mg/kgweekly. In some methods, two or more monoclonal antibodies withdifferent binding specificities are administered simultaneously, inwhich case the dosage of each antibody administered falls within theranges indicated. Progress can be monitored by periodic assessment.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives can also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. Furthermore, the pharmaceutical composition of theinvention can comprise further agents such as dopamine orpsychopharmacologic drugs, depending on the intended use of thepharmaceutical composition.

Furthermore, in a particular embodiment of the invention thepharmaceutical composition can be formulated as a vaccine, for example,if the pharmaceutical composition of the invention comprises ananti-TDP-43 antibody or binding fragment, derivative or variant thereoffor passive immunization. It is prudent to expect that the human antiTDP-43 antibodies and equivalent TDP-43 binding molecules of theinvention are particularly useful as a vaccine for the prevention oramelioration of TDP-43 proteinopathies such as amyotrophic lateralsclerosis (ALS) argyrophilic grain disease, Alzheimer's disease,ALS-Parkinsonism dementia complex of Guam, corticobasal degeneration,Dementia with Lewy bodies, Huntington's disease, Lewy body disease,motor neuron disease, frontotemporal lobar degeneration (FTLD),frontotemporal dementia, frontotemporal lobar degeneration withubiquitin-positive inclusions, hippocampal sclerosis, inclusion bodymyopathy, inclusion body myositis, Parkinson's disease, Parkinson'sdisease dementia, Parkinson-dementia complex in Kii peninsula, Pick'sdisease, Machado-Joseph disease and the like.

In one embodiment, it can be beneficial to use recombinant Fab (rFab)and single chain fragments (scFvs) of the antibody of the invention,which might more readily penetrate a cell membrane. For example, Robertet al., Protein Eng. Des. Sel. (2008) Oct. 16; S1741-0134, publishedonline ahead, describe the use of chimeric recombinant Fab (rFab) andsingle chain fragments (scFvs) of monoclonal antibody WO-2 whichrecognizes an epitope in the N-terminal region of Aβ. The engineeredfragments were able to (i) prevent amyloid fibrillization, (ii)disaggregate preformed Aβ1-42 fibrils and (iii) inhibit Aβ1-42oligomer-mediated neurotoxicity in vitro as efficiently as the whole IgGmolecule. The perceived advantages of using small Fab and scFvengineered antibody formats which lack the effector function includemore efficient passage across the blood-brain barrier and minimizing therisk of triggering inflammatory side reactions. Furthermore, besidesscFv and single-domain antibodies retain the binding specificity offull-length antibodies, they can be expressed as single genes andintracellularly in mammalian cells as intrabodies, with the potentialfor alteration of the folding, interactions, modifications, orsubcellular localization of their targets; see for review, e.g., Millerand Messer, Molecular Therapy 12 (2005), 394-401.

In a different approach Muller et al., Expert Opin. Biol. Ther. (2005),237-241, describe a technology platform, so-called ‘SuperAntibodyTechnology’, which is said to enable antibodies to be shuttled intoliving cells without harming them. Such cell-penetrating antibodies opennew diagnostic and therapeutic windows. The term ‘TransMabs’ has beencoined for these antibodies.

In a further embodiment, co-administration or sequential administrationof other neuroprotective agents useful for treating a TDP-43proteinopathy can be desirable. In one embodiment, the additional agentis comprised in the pharmaceutical composition of the invention.Examples of neuroprotective agents which can be used to treat a subjectinclude, but are not limited to, an acetylcholinesterase inhibitor, aglutamatergic receptor antagonist, kinase inhibitors, HDAC inhibitors,anti-inflammatory agents, divalproex sodium, or any combination thereof.Examples of other neuroprotective agents that can be used concomitantwith pharmaceutical composition of the invention are described in theart; see, e.g. International Application Publication No. WO2007/011907.In one embodiment, the additional agent is dopamine or a dopaminereceptor agonist.

A therapeutically effective dose or amount refers to that amount of theactive ingredient sufficient to ameliorate the symptoms or condition.Therapeutic efficacy and toxicity of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED₅₀ (the dose therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose lethal to 50% of the population). Thedose ratio between therapeutic and toxic effects is the therapeuticindex, and it can be expressed as the ratio, LD₅₀/ED₅₀. In oneembodiment, the therapeutic agent in the composition is present in anamount sufficient to restore or preserve normal behavior and/orcognitive properties in case of ALS and/or FTLD or other TDP-43proteinopathies.

From the foregoing, it is evident that the invention encompasses any useof an TDP-43 binding molecule comprising at least one CDR of the abovedescribed antibody, in particular for diagnosing and/or treatment of aTDP-43 proteinopathies as mentioned above, particularly amyotrophiclateral sclerosis and/or frontotemporal lobar degeneration. In oneembodiment, said binding molecule is an antibody of the invention or animmunoglobulin chain thereof. In addition, the invention relates toanti-idiotypic antibodies of any one of the mentioned antibodiesdescribed hereinbefore. Anti-idiotypic antibodies are antibodies orother binding molecules which bind to the unique antigenic peptidesequence located on an antibody's variable region near theantigen-binding site and are useful, e.g., for the detection ofanti-TDP-43 antibodies in sample of a subject.

In another embodiment the invention relates to a diagnostic compositioncomprising any one of the above described TDP-43 binding molecules,antibodies, antigen-binding fragments, polynucleotides, vectors or cellsof the invention and optionally suitable means for detection such asreagents conventionally used in immuno or nucleic acid based diagnosticmethods. The antibodies of the invention are, for example, suited foruse in immunoassays in which they can be utilized in liquid phase orbound to a solid phase carrier. Examples of immunoassays which canutilize the antibody of the invention are competitive andnon-competitive immunoassays in either a direct or indirect format.Examples of such immunoassays are the radioimmunoassay (RIA), thesandwich (immunometric assay), flow cytometry and the Western blotassay. The antigens and antibodies of the invention can be bound to manydifferent carriers and used to isolate cells specifically bound thereto.Examples of known carriers include glass, polystyrene, polyvinylchloride, polypropylene, polyethylene, polycarbonate, dextran, nylon,amyloses, natural and modified celluloses, polyacrylamides, agaroses,and magnetite. The nature of the carrier can be either soluble orinsoluble for the purposes of the invention. There are many differentlabels and methods of labeling known to those of ordinary skill in theart. Examples of the types of labels which can be used in the inventioninclude enzymes, radioisotopes, colloidal metals, fluorescent compounds,chemiluminescent compounds, and bioluminescent compounds; see also theembodiments discussed hereinabove.

By a further embodiment, the TDP-43 binding molecules, in particularantibodies of the invention can also be used in a method for thediagnosis of a disorder in an individual by obtaining a body fluidsample from the tested individual which can be a blood sample, a lymphsample or any other body fluid sample and contacting the body fluidsample with an antibody of the instant invention under conditionsenabling the formation of antibody-antigen complexes. The level of suchcomplexes is then determined by methods known in the art, a levelsignificantly higher than that formed in a control sample indicating thedisease in the tested individual. In the same manner, the specificantigen bound by the antibodies of the invention can also be used. Thus,the invention relates to an in vitro immunoassay comprising the bindingmolecule, e.g., antibody or antigen-binding fragment thereof of theinvention.

In this context, the invention also relates to means specificallydesigned for this purpose. For example, an antibody-based array can beused, which is for example loaded with antibodies or equivalentantigen-binding molecules of the invention which specifically recognizeTDP-43. Design of microarray immunoassays is summarized in Kusnezow etal., Mol. Cell Proteomics 5 (2006), 1681-1696. Accordingly, theinvention also relates to microarrays loaded with TDP-43 bindingmolecules identified in accordance with the invention.

In one embodiment, the invention relates to a method of diagnosing aTDP-43 proteinopathy in a subject, the method comprising:

-   -   (a) assessing the level of TDP-43 in a sample from the subject        to be diagnosed with an antibody of the invention, an TDP-43        binding fragment thereof or an TDP-43 binding molecule having        substantially the same binding specificities of any one thereof;        and    -   (b) Comparing the level of the TDP-43 to a reference standard        that indicates the level of the TDP-43 in one or more control        subjects,    -   Wherein a difference or similarity between the level of the        TDP-43 and the reference standard indicates that the subject        suffers from a TDP-43 proteinopathy.

The subject to be diagnosed can be asymptomatic or preclinical for thedisease. In one embodiment, the control subject has a TDP-43proteinopathy, for example ALS or FTLD, wherein a similarity between thelevel of TDP-43 and the reference standard indicates that the subject tobe diagnosed has a TDP-43 proteinopathy. Alternatively, or in additionas a second control the control subject does not have a TDP-43proteinopathy, wherein a difference between the level of TDP-43 and thereference standard indicates that the subject to be diagnosed has aTDP-43 proteinopathy. I, the subject to be diagnosed and the controlsubject(s) are age-matched. The sample to be analyzed can be any bodyfluid suspected to contain TDP-43, for example a blood, CSF, or urinesample.

The level of TDP-43 can be assessed by any suitable method known in theart comprising, e.g., analyzing TDP-43 by one or more techniques chosenfrom Western blot, immunoprecipitation, enzyme-linked immunosorbentassay (ELISA), radioimmunoassay (RIA), fluorescent activated cellsorting (FACS), two-dimensional gel electrophoresis, mass spectroscopy(MS), matrix-assisted laser desorption/ionization-time of flight-MS(MALDI-TOF), surface-enhanced laser desorption ionization-time of flight(SELDI-TOF), high performance liquid chromatography (HPLC), fast proteinliquid chromatography (FPLC), multidimensional liquid chromatography(LC) followed by tandem mass spectrometry (MS/MS), and laserdensitometry. In one embodiment, said in vivo imaging of TDP-43comprises positron emission tomography (PET), single photon emissiontomography (SPECT), near infrared (NIR) optical imaging or magneticresonance imaging (MRI).

Methods of diagnosing a neurodegenerative disease such as AD,Parkinson's disease, ALS, Huntington's disease, Dementia with Lewybodies or FTLD for monitoring a TDP-43 proteinopathy progression, andmonitoring a TDP-43 proteinopathy treatment using antibodies and relatedmeans which can be adapted in accordance with the invention are alsodescribed in International Application Publication Nos. WO 2010/111587and WO2007/011907, which is herein incorporated by reference in itsentirety. Those methods can be applied as described but with an TDP-43specific antibody, binding fragment, derivative or variant of theinvention.

These and other embodiments are disclosed and encompassed by thedescription and examples of the invention. Further literature concerningany one of the materials, methods, uses and compounds to be employed inaccordance with the invention can be retrieved from public libraries anddatabases, using for example electronic devices. For example the publicdatabase “Medline” can be utilized, which is hosted by the NationalCenter for Biotechnology Information and/or the National Library ofMedicine at the National Institutes of Health. Further databases and webaddresses, such as those of the European Bioinformatics Institute (EBI),which is part of the European Molecular Biology Laboratory (EMBL) areknown to the person of ordinary skill in the art and can also beobtained using internet search engines. An overview of patentinformation in biotechnology and a survey of relevant sources of patentinformation useful for retrospective searching and for current awarenessare given in Berks, TIBTECH 12 (1994), 352-364.

The above disclosure generally describes the invention. Unless otherwisestated, a term as used herein is given the definition as provided in theOxford Dictionary of Biochemistry and Molecular Biology, OxfordUniversity Press, 1997, revised 2000 and reprinted 2003, ISBN 0 19850673 2. Several documents are cited throughout the text of thisspecification. Full bibliographic citations can be found at the end ofthe specification immediately preceding the claims. The contents of allcited references (including literature references, issued patents,published patent applications as cited throughout this application andmanufacturer's specifications, instructions, etc.) are herebyincorporated by reference in their entireties; however, there is noadmission that any document cited is indeed prior art as to theinvention.

A more complete understanding can be obtained by reference to thefollowing specific examples which are provided herein for purposes ofillustration only and are not intended to limit the scope of theinvention.

EXAMPLES Material and Methods

Detailed descriptions of conventional methods, such as those employedherein can be found in the cited literature. Unless indicated otherwisebelow, identification of TDP-43-specific B cells and molecular cloningof TDP-43 antibodies displaying specificity of interest as well as theirrecombinant expression and functional characterization has been or canbe performed as described in the Examples and Supplementary Methodssection of International Application PCT/EP2008/000053 published asWO2008/081008, each of which are herein incorporated by reference in itsentirety.

Human TDP-43 Antibody Screening

ELISA

96 well half area microplates (Corning) were coated with either:

-   -   (a) recombinant full-length His-tagged human TDP-43 (Biogen        Idec, USA); or    -   (b) a synthetic peptide consisting of residues 390-414 of the        C-terminal domain of TDP-43 with phosphorylation modification at        residues 409 and 410 (Shafer-N, DK)    -   at a concentration of 5 μg/ml and 3.3 μg/ml, respectively, in        carbonate ELISA coating buffer (pH 9.6) overnight at 4° C.

Plates were washed in PBS TWEEN® (pH 7.6) and non-specific binding siteswere blocked for 1 hr. at room temperature with PBS-TWEEN® containing 2%BSA (Sigma, Buchs, Switzerland). B cell conditioned medium wastransferred from memory B cell culture plates to ELISA plates andincubated for 1 hr. at room temperature. ELISA plates were washed inPBS-TWEEN® and then incubated with horse radish peroxidase(HRP)-conjugated anti-human immunoglobulins polyclonal antibodies(Jackson ImmunoResearch, USA). After washing with PBS-TWEEN®, binding ofhuman antibodies was determined by measurement of HRP activity in astandard colorimetric assay.

MSD

Standard 96 well 10-Spot MULTI-SPOT plates (Meso Scale Discovery, USA)were coated with a mixture of TDP-43 protein fragments corresponding toamino acids 1-259, 260 to 277 and 350-366 (Abcam plc, UK), respectively.10 μg/ml of each peptide was used and formulated in PBS. Non-specificbinding sites were blocked for 1 hr. at room temperature with PBS-TWEEN®containing 3% BSA followed by incubation with B cell conditioned mediumfor 1 hr. at room temperature. Plates were washed in PBS-TWEEN® and thenincubated with SULFO-Tag conjugated anti-human polyclonal antibody(Mesoscale Discovery, USA). After washing with PBS-TWEEN®, boundantibody was detected by electrochemiluminescence measurement using aSECTOR Imager 6000 (Meso Scale Discovery, USA).

Molecular Cloning of Human TDP-43 Antibodies

Samples containing memory B cells were obtained from healthy humansubjects. Living B cells of selected memory B cell cultures wereharvested, mRNA was isolated and cDNA was prepared by ReverseTranscriptase (Clontech, USA). Immunoglobulin heavy and light chainsequences were then obtained using a nested PCR approach.

A combination of primers representing all sequence families of the humanimmunoglobulin germline repertoire are used for the amplifications ofleader peptides, V-segments and J-segments. The first roundamplification is performed using leader peptide-specific primers in5′-end and constant region-specific primers in 3′-end (Smith et al., NatProtoc. 4 (2009), 372-384). For heavy chains and kappa light chains, thesecond round amplification is performed using V-segment-specific primersat the 5′-end and J-segment-specific primers at the 3′ end. For lambdalight chains, the second round amplification is performed usingV-segment-specific primers at the 5′-end and a C-region-specific primerat the 3′ end (Marks et al., Mol. Biol. 222 (1991), 581-597; de Haard etal., J. Biol. Chem. 26 (1999), 18218-18230).

Identification of the antibody clone with the desired specificity isperformed by re-screening on ELISA upon recombinant expression ofcomplete antibodies.

Recombinant expression of complete human IgG1 antibodies or chimericIgG2a antibodies is achieved upon insertion of the variable heavy andlight chain sequences “in the correct reading frame” into expressionvectors that complement the variable region sequence with a sequenceencoding a leader peptide at the 5′-end and with a sequence encoding theappropriate constant domain(s) at the 3′-end. To that end the primerscontained restriction sites designed to facilitate cloning of thevariable heavy and light chain sequences into antibody expressionvectors. Heavy chain immunoglobulins are expressed by inserting theimmunoglobulin heavy chain RT-PCR product in frame into a heavy chainexpression vector bearing a signal peptide and the constant domains ofhuman immunoglobulin gamma 1 or mouse immunoglobulin gamma 2a. Kappalight chain immunoglobulins are expressed by inserting the kappa lightchain RT-PCR-product in frame into a light chain expression vectorproviding a signal peptide and the constant domain of human kappa lightchain immunoglobulin. Lambda light chain immunoglobulins are expressedby inserting the lambda light chain RT-PCR-product in frame into alambda light chain expression vector providing a signal peptide and theconstant domain of human or mouse lambda light chain immunoglobulin.

Functional recombinant monoclonal antibodies are obtained uponco-transfection into HEK293 or CHO cells (or any other appropriaterecipient cell line of human or mouse origin) of an Ig-heavy-chainexpression vector and a kappa or lambda Ig-light-chain expressionvector. Recombinant human monoclonal antibody is subsequently purifiedfrom the conditioned medium using a standard Protein A columnpurification. Recombinant human monoclonal antibody can be produced inunlimited quantities using either transiently or stably transfectedcells. Cell lines producing recombinant human monoclonal antibody can beestablished either by using the Ig-expression vectors directly or byre-cloning of Ig-variable regions into different expression vectors.Derivatives such as F(ab), F(ab)2 and scFv can also be generated fromthese Ig-variable regions.

Purification and Characterization of his-Tagged TDP-43

An expression vector for 6×His-tagged TDP-43 (pCGB026) was generated bycloning the TDP-43 sequence into the pRSET A expression vector(INVITROGEN™). BL21 STAR™ (DE3)pLysS E. coli cells (INVITROGEN™) weretransformed with pCGB026. Following growth at 37° C. to approximately 1OD in a 1 L shake flask containing LB broth, IPTG was added to 0.5 mMand the cells were grown overnight at 18° C. Cells were pelleted bylow-speed centrifugation, the supernatant was decanted and the cellpellets were frozen for storage at −20° C.

Cell pellets were equilibrated to room temperature and resuspended in 50mL of 50 mM Tris-HCl pH 7.5, 20 mM imidazole, 150 mM NaCl, containingprotease inhibitors (PI) (final concentration PI: 1 mM PMSF, 5 μMpepstatin A, 1 mM benzamide, 10 μM bestatin, 10 μM E64, 20 μM leupeptin,1.5 μM aprotinin). Following homogenization for 5 min. to break up largeparticles, the cells were disrupted under high pressure using aMicrofluidizer (Microfluidics, Inc.). The cell lysate was centrifugedfor 30 min. at 10,000 rpm at 4° C. The pellet, containing insolublehis-tagged TDP-43, was resuspended in 5 mL B-PER solution (ThermoScientific) containing 2 mM MgCl₂, COMPLETE™ EDTA-free PI tablets(Roche), 100 μg/mL lysozyme, 5 U/mL DNase (Thermo Scientific) andincubated for 15 min. at room temperature. Additional B-PER/2 mMMgCl₂/PI tablet solution was added to a final volume of 50 mL and themixture was centrifuged for 10 min. at 10,000 rpm. The isolated pelletwas washed twice in 20 mL of 50 mM Tris-HCl pH 7.5, 20 mM imidazole, 150mM NaCl, PI buffer, followed by centrifugation for 10 min. at 10,000rpm. The isolated pellet was then resuspended in 8 M urea, 20 mM sodiumphosphate pH 7.8 containing PI, mixed for 5 min. by homogenization andcentrifuged at 10,000 rpm for 10 min.

The supernatant was isolated, filtered through a 0.45 μm filter,combined with 4 mL of Ni-NTA resin (Invitrogen) equilibrated in 8 Murea, 20 mM sodium phosphate pH 7.8, 0.5 M NaCl, with PI, and incubatedwhile rocking overnight at 4° C. The flow-through was collected and theresin was washed with 10 column volumes of 8 M urea, 20 mM sodiumphosphate pH 5.3, 0.5 M NaCl. His-tagged TDP-43 was eluted in 0.5 columnvolume fractions with 8 M urea, 20 mM sodium phosphate pH 4, 0.5 Msodium chloride. Peak fractions were pooled, and protein concentrationwas determined using UV spectrometry. The total purified yield was ˜8mg/L culture.

SDS-PAGE analysis of the purified protein showed a major band at ˜47 kDa(data not provided), consistent with the predicted molecular mass of46.5 kDa. Intact mass spectrometry on the purified protein indicated amajor peak at 46538 Da, consistent with the predicted mass of 46529.9Da. The ability of known commercially available antibodies againstTDP-43 to bind the purified protein was determined by ELISA. Mousemonoclonal antibody 2E2-D3, recognizing amino acids 205-222 (Zhang,H.-X. et al., 2008, Neuroscience Lett., 434, 170-74), and rabbitpolyclonal antibodies A260 and G400, recognizing sequences around aminoacids Ala-260 and Gly-400, respectively, all bound to the purifiedHis-tagged TDP-43 (data not provided).

Recombinant Expression of Human TDP-43 Domains

The human TDP-43 domain coding sequences were amplified by PCR from thefull length cDNA sequence (Q13148, TARDBP_HUMAN) and cloned into theexpression vector pRSET-A (INVITROGEN™, USA). The four expressionvectors (His-huTDP-43 domain I, His-huTDP-43 domain II, His-huTDP-43domain III and His-huTDP-43 domain IV) are coding for the four TDP-43domains: the N-terminal domain (amino acid residues 2-106 of SEQ IDNO:94), the RNA binding domain 1 (amino acid residues 99-204 of SEQ IDNO:94), the RNA binding domain 2 (amino acid residues 183-273 of SEQ IDNO:94) and the Glycine-rich domain (amino acid residues 258-414 of SEQID NO:94), respectively. DNA constructs comprising the cDNA encoding theTDP-43 domains under the control of the T7 promoter were used totransform an appropriate Escherichia coli strain such as BL21 (DE3) (NewEngland Biolabs, USA) and expression of 15 ml cell culture was inducedby the addition of 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG).Cells were harvested after 4 hrs. induction at 37° C. and thenresuspended in 1 ml 100 mM KCl, 50 mM HEPES, 2 mM EGTA, 1 mM MgCl₂, 1 mMDithiothreitol, 0.1 mM PMSF, 10% glycerol and 0.1 mg/ml lysozyme, pH7.5, followed by sonification. Soluble and insoluble fractions werecollected after centrifugation at 9000 rpm at 4° C. for 45 min.Similarly, 9000 g supernatant from mock Escherichia coli was collected.When required (TDP-43 domain IV), insoluble fraction was solubilized in1 ml 8M Urea, 20 mM Tris, 200 mM KCl and 1 mM β-Mercaptoethanol. Solubleand solubilized fractions were loaded onto Ni-NTA SUPERFLOW™ Columns(QIAGEN®, USA) and His-TDP-43 domains were purified according tomanufacturer's protocol. Purity grade of recombinant proteins wasestimated by SDS-PAGE and Coomassie staining. Concentration of purifiedproteins was determined by 280 nM absorbance measurement.

Direct ELISA

96 well microplates (Corning) were coated with human full length TDP-43,TDP-43 domain I (amino acid residues 2-106 of SEQ ID NO:94), TDP-43domain II (amino acid residues 99-204 of SEQ ID NO:94), TDP-43 domainIII (amino acid residues 183-273 of SEQ ID NO:94), TDP-43 domain IV(amino acid residues 258-414 of SEQ ID NO:94) or with a syntheticpeptide covering residues 390 to 414 of the C-terminal domain of TDP-43(see, SEQ ID NO:94) with phosphorylation modification at residues409/410 (Schafer-N, DK) diluted to a concentration of 6.6 μg/ml or 3.3μg/ml, respectively, in carbonate ELISA coating buffer (pH 9.6)overnight at 4° C. Non-specific binding sites were blocked for 1 hr. atroom temperature with PBST containing 2% BSA (Sigma, Buchs,Switzerland). Antibodies were incubated 1 hr. at room temperature.Binding was determined using either a donkey anti-human IgGγ-specificantibody conjugated with HRP (Jackson ImmunoResearch, USA) or agoat-anti mouse IgG (H+L)-specific secondary antibody conjugated withHRP (Jackson ImmunoResearch, USA)., followed by measurement of HRPactivity in a standard colorimetric assay.

Western Blot Analysis

Recombinant full length TDP-43, TDP-43 domain I (amino acid residues2-106 of SEQ ID NO:94), TDP-43 domain II (amino acid residues 99-204 ofSEQ ID NO:94), TDP-43 domain III (amino acid residues 183-273 of SEQ IDNO:94), TDP-43 domain IV (amino acid residues 258-414 of SEQ ID NO:94),300 ng of each, were resolved by SDS-PAGE (NUPAGE® 12% Bis-Tris Gel;INVITROGEN™, Basel, Switzerland) followed by electroblotting onnitrocellulose membranes. Non-specific binding sites were blocked for 1hr. at room temperature with PBST containing 2% BSA (Sigma, Buchs,Switzerland). Blots were incubated overnight with primary antibodies (10nM) followed by either a donkey anti-human IgGy-specific secondaryantibody conjugated with HRP (Jackson ImmunoResearch, USA) or agoat-anti mouse IgG (H+L)-specific secondary antibody conjugated withHRP (Jackson ImmunoResearch, USA). Blots were developed using ECL andImageQuant 350 detection (GE Healthcare, Otelfingen, Switzerland).

Two-Trial Y-Maze Task

Improvement of working memory in antibody treated TDP-43 proteinopathymouse model can be tested using a two-trial Y-maze task (e.g., Pennanen,Genes Brain Behay. 5 (2006), 369-79, which is herein incorporated byreference in its entirety). The three arms of the maze are 22 cm long, 5cm wide and 15 cm deep. Black and white abstractive clues are placed ona black curtain surrounding the maze. Experiments are conducted with anambient light level of 6 lux during the dark phase. Each experimentcomprises a training session and an observation session. During thetraining session, a mouse is assigned to two of the three arms (thestart arm and the second arm), which can be freely explored during 4min, with no access to the third arm (the novel arm). The mouse is thenremoved from the maze and kept in a holding cage for 1.5-5 min, whilethe maze is thoroughly cleaned with 70% ethanol to remove any olfactoryclues. The mouse is then put back again in the maze for observation withall three arms accessible for 4 min. The sequence of entries, the numberof entry to each arm and the time spent in each arm is recorded. Fromthat the ratio of time spent in the novel third arm over the average oftime spent in the other two arms (start arm and second arm) iscalculated and compared among different treatment groups in tauopathymouse model and corresponding control wild type mice. Rodents typicallyprefer to investigate a new arm of the maze rather than returning to onethat was previously visited. Effects of the antibodies can be monitoredin regard of regaining this preference by treated TDP-43 proteinopathymodel mice in comparison to non-discriminative behavior of untreatedmice due to their disorder-related working memory impairment. Therefore,a ratio close to 1 indicates impaired working memory. A higher ratioindicates better working memory. Impaired working memory in a TDP-43proteinopathy model mice is considered to be due to TDP-43 pathologyresulting from the overexpression of human TDP-43. Therefore asignificantly higher ratio observed in anti-TDP-43 antibody treated micethan in the control mice will indicate that the anti-TDP-43 antibody hastherapeutic effect on TDP-43 pathology.

Pole Test

Mice are tested at the beginning of the dark phase when they are mostactive. The pole is made of a wooden stick with 50 cm length and 1 cmwidth covered with cloth to facilitate climbing. The base of the pole isplaced in the home cage of the mouse. The mouse is placed on the top ofthe pole and the time to orient downwards and time to climb down to thehome cage is recorded over 5 trials with 30 min intertrial intervals.The best performance trial is analyzed.

Elevated Plus Maze Test

Mice are tested at the beginning of the dark phase when they are mostactive. Testing is performed in dim light (40 lux). The elevated plusmaze consists of two open and two closed arms (arm length: 30 cm; width:5 cm). Open arms have a small 1 cm edge and the closed arms are borderedby a 15 cm wall. At the beginning of the task, mice are placed in thecenter of the elevated plus maze facing an open arm. Mice arevideo-tracked while exploring the maze for 5 min. The time spent in theopen and closed arms and the distance covered are measured and analyzed.

Example 1: Human TDP-43 Antibody Binding Analysis by ELISA and WesternBlotting

Direct ELISAs

96 well microplates (Corning) were coated with polypeptides comprisingTDP-43 amino acid residues 2-106 of SEQ ID NO:94 (TDP-43 domain I),residues 99-204 of SEQ ID NO:94 (TDP-43 domain II), residues 183-273 ofSEQ ID NO:94 (TDP-43 domain III), residues 258-414 of SEQ ID NO:94(TDP-43 domain IV), residues 2-414 of SEQ ID NO:94 (full-length TDP-43)and a synthetic polypeptide containing residues 390-414 withphosphorylation modification at residues 409/410 of SEQ ID NO:94. Thepolypeptides were coated onto ELISA plates at equal coatingconcentration of 6.6 μg/ml (recombinant TDP-43 and TDP-43 fragments) or3.3 μg/ml (synthetic peptides). Binding of the human-derived antibodieswas determined by direct ELISA.

Antibodies NI-205.51C1 (FIG. 2A) and NI-205.3F10 (FIG. 2C) boundspecifically to TDP-43 domain III (amino acid residues 183-273 of SEQ IDNO:94). Antibodies NI-205.8A2 (FIG. 2D), NI-205.15F12 (FIG. 2E),NI-205.25F3 (FIG. 2G) and NI-205.21G1 (FIG. 2I) bound specifically toTDP-43 domain IV (amino acid residues 258-414 of SEQ ID NO:94). AntibodyNI-205.87E7 (FIG. 2H) bound specifically to TDP-43 domain I (amino acidresidues 2-106 of SEQ ID NO:94). Antibodies NI-205.21G2 (FIG. 2B) andNI-205.113C4 (FIG. 2F) bound specifically to TDP-43 domain II (aminoacid residues 99-204 of SEQ ID NO:94). All human derived TDP-43 specificantibodies specifically recognized full-length TDP-43. To control forcoating efficiency, of the different TDP-43 domains, commerciallyavailable antibodies binding to full-length TDP-43 and a specific TDP-43domain were used: Ab50930 (Abcam, UK), TDP-43 domain-1; TARDBPmonoclonal antibody (M01), clone 2E2-D3 (Abnova, Taiwan), TDP-43 domainIII, and Ab82695 (Abcam, US), TDP-43 domain IV. Control antibodies boundto full-length TDP-43 and their specific TDP-43 domain.

Binding to Distinct TDP-43 Domains by Western Blot

Recombinant full length TDP-43, TDP-43 domain I (amino acid residues2-106 of SEQ ID NO:94), TDP-43 domain II (amino acid residues 99-204 ofSEQ ID NO:94), TDP-43 domain III (amino acid residues 183-273 of SEQ IDNO:94), and TDP-43 domain IV (amino acid residues 258-414 of SEQ IDNO:94) were resolved by SDS-PAGE. Commercially available TDP-43 specificantibody TARDBP (M01), clone 2E2-D3 (Abnova, Taiwan) was used aspositive control for human TDP-43 detection whereas, an anti-human IgGFcγ-specific antibody was used as a negative control. Binding of thehuman-derived antibodies to specific TDP-43 domains was determined byWestern Blot analysis.

Western blotting indicated that antibodies: (a) NI-205.51C1 andNI-205.3F10 antibodies bound specifically to TDP-43 domain III (aminoacid residues 183-273 of SEQ ID NO:94); (b) NI-205.8A2 and NI-205.21G1bound specifically to TDP-43 domain IV (amino acid residues 258-414 ofSEQ ID NO:94); (c) NI-205.21G2 specifically recognized TDP-43 domain II(amino acid residues 99-204 of SEQ ID NO:94); (d) NI-205.51C1,NI-205.3F10, NI-205.8A2, NI-205.21G1, NI-205.21G2, recognized fulllength human TDP-43 in addition to a domain of TDP-43; and (e)human-derived antibodies NI-205.25F3, NI-205.15F12 and NI-205.87E7recognized full length TDP-43 but did not appear to bind a specificTDP-43 domain (data not provided). Additionally, the antibodiesNI-205.68G5 and NI-205.20A1, that preferentially or exclusively boundthe phospho-TDP-43 C-terminal peptide (see Example 2), did not appear torecognize recombinant full length TDP-43 or any of its fragments (datanot provided).

Example 2: EC₅₀ Determination for Human TDP-43 Binding AntibodiesDetermination of Half Maximal Effective Concentration (EC₅₀)

To determine the half maximal effective concentration (EC₅₀) of thehuman-derived TDP-43-specific antibodies for human TDP-43 and toevaluate target-specificity, 96 well microplates (Corning) were coatedwith recombinant full length TDP-43 (Biogen Idec, USA), Escherichia coliextract and BSA (Sigma, Buchs, Switzerland), diluted to a concentrationof 5 μg/ml in carbonate ELISA coating buffer (pH 9.6) overnight at 4° C.Alternatively, 96 well half area microplates (Corning) were coated witha synthetic peptide covering residues 390 to 414 of the C-terminaldomain of TDP-43 with phosphorylation modification at residues 409/410(Schafer-N, DK) and BSA (Sigma, Buchs, Switzerland), diluted to aconcentration of 3.3 μg/ml in carbonate ELISA coating buffer (pH 9.6)overnight at 4° C. Non-specific binding sites were blocked for 1 hr. atroom temperature with PBS containing 2% BSA (Sigma, Buchs, Switzerland).Human TDP-43-specific antibodies were diluted to the indicatedconcentrations and incubated 1 hr. at room temperature. Binding wasdetermined using a donkey anti-human IgGγ-specific secondary antibodyconjugated with HRP (Jackson ImmunoResearch, USA), followed bymeasurement of HRP activity in a standard colorimetric assay. EC₅₀values were estimated by a non-linear regression using GraphPad Prismsoftware (San Diego, USA).

As disclosed in Table 4, antibodies NI-205.51C1 and NI-205.21G2 bind tohuman TDP-43 with high affinity at subnanomolar EC₅₀ of 180 pM and 240pM, respectively. No binding was observed to the phospho-TDP-43C-terminal peptide for these antibodies. Antibodies NI-205.3F10,NI-205.8A2, NI-205.15F12, NI-205.113C4, NI-205.25F3, and NI-205.87E7bound to human TDP-43, but not to the phospho-TDP-43 C-terminal peptide.The EC₅₀ values of these antibodies for human TDP-43 proteins rangedfrom 1 to 18 nM. NI-205.21G1 bound to full-length TDP-43 at 4.1 nM EC₅₀and recognized phosphor-TDP-43 C-terminal peptide with lower affinity at49.5 nM EC₅₀. Antibodies NI-205.68G5 and NI-205.20A1 showed preferentialor exclusive binding to the phosphor-TDP-43 C-terminal peptide at 16.9and 15.8 nM EC₅₀, respectively, suggesting that phosphorylation ofserine at position 409 and/or serine at position 410 of TDP-43 isrequired for the binding by NI-205.68G5 and NI-205.20A1.

TABLE 4 Antibody Affinity for TDP-43 and phosphorylated TDP-43Full-length TDP-43 Phospho-TDP-43 Peptide Antibody EC₅₀ (nM)] EC₅₀ (nM)NI-205.51C1 0.18 N.A. NI-205.21G2 0.24 N.A. NI-205.3F10 1.4-2.8 N.A.NI-205.8A2 7.2 N.A. NI-205.15F12 7.2 N.A. NI-205.113C4 13.2 N.A.NI-205.25F3 13.3 N.A. NI-205.87E7 17.2 N.A. NI-205.21G1 4.1 49.5NI-205.68G5 >100 16.9 NI-205.20A1 N.A. 15.8 Table 4: EC₅₀ binding ofhuman derived TDP-43 binding antibodies to recombinant TDP-43 andphosphor-TDP-43 peptide.

Example 3: Epitope Mapping with Synthetic Peptides (PepSpotting)

Scans of overlapping peptides were used to map the epitopes within thehuman TDP-43 protein that are recognized by the human-derived TDP-43specific antibodies. Pepscan membranes (PEPSPOT™, JPT PeptideTechnologies, Berlin, Germany) with 101 linear 15mer peptides of 11amino acid overlapping sequences that collectively represent the entiresequence of human TDP-43 (Q13148, TARDBP_HUMAN). The peptides werespotted onto nitrocellulose membranes that were then activated for 5min. in methanol and subsequently washed at room temperature in TBS for10 min. Non-specific binding sites were blocked for 2 hours at roomtemperature with ROTI®-Block (Carl Roth GmbH Co. KG, Karlsruhe,Germany). Human-derived TDP-43 specific antibodies (1 μg/ml) wereincubated for 3 hours at room temperature in ROTI®-Block. Binding ofprimary antibody was determined using HRP conjugated donkey-anti humanIgGγ-specific secondary antibody (Jackson ImmunoResearch, USA). Blotswere developed using ECL and ImageQuant 350 detection (GE Healthcare,Otelfingen, Switzerland).

Table 4 summarizes the identified binding epitopes for the differenthuman-derived TDP-43-specific antibodies identified using PEPSPOT™.

TABLE 5 Identified binding epitopes within the humanTDP-43 protein sequence Antibody Binding epitope NI-205.3F10213-QYGDVMDVFIP-223 (SEQ ID NO: 123) NI-205.8A2381-AAIGWGSASNA-391 (SEQ ID NO: 124) NI-205.51C1201-DMTEDELREFF-211 (SEQ ID NO: 125) NI-205.87E79-EDENDEP-15 (SEQ ID NO: 126) NI-205.113C4133-VQVKKDL-139 (SEQ ID NO: 127) NI-205.21G2121-KEYFSTF-127 (SEQ ID NO: 128)

Example 4: Binding of Human Recombinant TDP-43 Antibodies to PathologicForms of TDP-43 in Human Spinal Cord and Brain Tissue

For validation of TDP-43 antibody-binding capacity, spinal cord andbrain sections derived from human ALS patients or patients with FTLDwere used. Antibody binding to TDP-43 pathology was assessed byimmunohistochemical staining. Binding of human recombinant TDP-43antibodies was characterized on human FTLD-TDP-43 case tissue (10patient cases, 7 control cases). Immunohistochemistry was performed onSum thick paraffin embedded sections, and included the use of EDTA-basedepitope retrieval prior to conducting otherwise standardimmunoperoxidase procedures with Elite ABC kits (Vector Laboratories)with DAB (Pierce). The following primary antibodies were used: mousemonoclonal antibody 2E2-D3 raised against human TDP-43 (Abnova) as apositive control; recombinant human TDP-43 antibodies describedhere/above NI205.3F10, NI205.51C1, NI205.21 G2, NI205.8A2, NI205.15F12,NI205.25F3, NI205.87E7, NI205.21G1, NI205.68G5, NI205.20A1. Sectionswere counterstained with Haematoxylin to reveal cell nuclei.

The antibodies NI205.8A2, NI205.3F10, NI205.21G2, and NI205.21G1preferentially bound to cytoplasmic TDP-43 (i.e., pathologic forms ofTDP-43) over nuclear TDP-43. By contrast, the commercially availablepositive control antibody, 2E2 (Abnova, Taiwan) was observed to bindboth nuclear and cytoplasmic TDP-43. Interestingly, the binding of theantibody NI205.21G1 demonstrated very specific binding that appears tobe comparable to the binding observed for control antibodies thatrecognize phosphorylated TDP-43 (FIGS. 11E and H).

Example 5: In Vivo Validation of TDP-43 Antibody

Experiments for preclinical validation of the TDP-43 antibodies areperformed in mouse models of TDP-43 proteinopathy. Human TDP-43antibodies are administered by peripheral injection or intraventricularbrain infusion via osmotic minipumps. Treatment effects are monitored byanalysis of blood and CSF samples, and analysis of body weight, generalclinical impression and signs of motor or cognitive impairment as seenthrough for example, though behavioral tests including open field,Y-maze, elevated plus maze, novel object recognition, grip strength, pawgrip strength endurance, pole test, challenging beam walk, rotarod, orotherwise known in the art.

Upon completion of the treatment studies, changes in TDP-43 levels incollected blood and CSF are measured and brain and spinal cord tissuesare evaluated by quantitative immunohistochemical and biochemicaltechniques for brain and spinal cord content of physiological andpathological TDP-43 and general neuropathology.

Preclinical models useful for validating the antibodies and other TDP-43binding molecules of the invention include the TDP-43-A315T mouse modelsystem as described by Wegorzewska et al., Proc. Natl. Acad. Sci. U.S.A.106 (2009), 18809-14. The A315T mouse is a transgenic model of TDP-43proteinopathy, wherein the phenotype of the mice shows features of bothALS and frontotemporal lobar degeneration with ubiquitin aggregates(FTLD-U).

Further suitable models include the B6.Cg-Tg (SOD1*G93A)1Gur/J mousemodel system as described by Gurney et al., Science 264 (1994), 1772-75.This mouse line expresses the G93A mutant form of the human superoxidedismutase 1 and develops signs of motor neuron disease followed byprogression to death within 6 to 8 months. These mice show acharacteristic redistribution of TDP-43 to the cytoplasm of motorneurons and the occurrence of TDP-43 immunoreactive inclusions and aretherefore a suitable model to study pharmacological interventionstargeting TDP-43; see e.g., Shan et al., Neuropharmacol. Letters 458(2009), 70-74.

Further experiments for validating the TDP-43 antibodies are performedin the TDP43WT mouse model system as described by Wils et al., Proc.Natl. Acad. Sci. USA. 106 (2010), 3858-63. This mouse line expresseswild type human TDP-43 and develops degeneration of cortical and spinalmotor neurons and development of spastic quadriplegia reminiscent ofALS. A dose-dependent degeneration of nonmotor cortical and subcorticalneurons characteristic of FTLD is also observed in this mouse line.Neurons in the affected spinal cord and brain regions show accumulationof TDP-43 nuclear and cytoplasmic aggregates that are both ubiquitinatedand phosphorylated as observed in ALS/FTLD patients.

Further experiments for validating the TDP-43 antibodies are performedin an independent model of motor neuron disease, the Wobbler mouse model(B6.B-Vps54wr/J, available from the Jackson Laboratories) as describedby Duchen and Strich, J. Neurol. Neurosurg. Psychiatry 31 (1968),535-42. This mouse model was reported to display extensive intracellularubiquitin inclusions and abnormal cytoplasmic distribution of TDP-43reminiscent to sporadic ALS, see e.g., Dennis and Citron, Neuroscience185 (2009), 745-50.

Further experiments for validating the TDP-43 antibodies are performedin recently characterized TDP-43_G348C transgenic mice overexpressinghuman genomic TDP-43_G348C under the control of the endogenous promoter.Swarup et al., Brain 134 (2011), 2610-2626.

Further experiments for validating the TDP-43 antibodies are performedin model systems, including transgenic cell lines and transgenicanimals, that express or overexpress TDP-43, TDP-43 mutants, such asC-terminal truncations of TDP-43, TDP-43 mutations seen in a patientpopulation, or mutants that effect cellular localization, e.g. nuclearlocalization of TDP-43. TDP-43 model systems, e.g., cell lines andanimal models, for validating the TDP-43 antibodies further includesystems with demonstrated TDP-43 upregulation or accumulation resultingfrom the changes in the expression of a different gene, e.g. the Wobblermouse and models with down-regulation of progranulin. In one embodiment,experiments for validating the TDP-43 antibodies are performed in miceexpressing human TDP-43 with a defective nuclear localization signal inthe forebrain (Igaz et al., J Clin Invest. 121(2):726-38 (2011));transgenic mice that selectively express 25-kDa C-terminal fragment ofTDP-43 in neurons (Caccamo et al., Am J Pathol. 180(1):293-302 (2012)),transgenic mice that conditionally express wild-type human TDP-43(hTDP-43) in the forebrain (Cannon et al., Acta Neuropathol.123(6):807-23 (2012)), and transgenic mice with ubiquitous expression ofwild-type and disease-causing versions of human VCP/p97 (Custer et al.,Hum Mol Genet. 19(9):1741-55 (2010)). In another embodiment, experimentsfor validating the TDP-43 antibodies are performed in mice transfectedwith an AAV vector encoding wild type TDP-43, nuclear localizationsignal defective TDP-43, or a truncated C-terminal fragment of TDP-43comprising residues 220-414 of SEQ ID NO: 94. Tatom et al., Mol. Ther.17 (2009), 607-613.

Chronic efficacy study: To assess the pharmacological effects of thehuman anti-TDP-43 antibodies disclosed herein, TDP-43_G348C transgenicmice (Swamp et al., Brain 134 (2011), 2610-2626) are treated weekly with10 mg/kg i.p. injection of a human anti-TDP-43 antibody or vehiclecontrol for a period of 16-24 weeks. After 12 weeks of treatment, bloodsamples are collected by tail vein bleeding. The serum anti-TDP-43antibody levels are determined by ELISA. After 12 weeks and 22 weeks oftreatment, neurological and cognitive/motor behavior is evaluated usingopen field test, Y-maze test, elevated plus maze test, novel objectrecognition test, grip strength test, paw grip strength endurance (PAGE)test, pole test, challenging beam walk test, or rotarod test. Theneurological and cognitive/motor behavior test results for antibodytreated and control animals are compared. Improved performance ofantibody treated animals indicates therapeutic efficacy of theanti-TDP-43 antibody.

Acute Efficacy Study:

TDP-43_G348C transgenic mice (Swarup et al., Brain 134 (2011),2610-2626) are treated with 1-4 i.p. injections of up to 50 mg/kganti-TDP-43 antibody or vehicle control within a period of one week. Atthe end of the treatment period, blood samples are collected by tailvein bleeding. The serum anti-TDP-43 antibody levels are determined byELISA. At the end of the 1-week treatment period, neurological andcognitive/motor behavior is evaluated using open field test, Y-mazetest, elevated plus maze test, novel object recognition test, gripstrength test, paw grip strength endurance (PAGE) test, pole test,challenging beam walk test, or rotarod test. The neurological andcognitive/motor behavior test results for antibody treated and controlanimals are compared. Improved performance of antibody treated animalsindicates therapeutic efficacy of the anti-TDP-43 antibody.

Example 6: Determination of Binding Affinity (EC₅₀) for Human TDP-43Antibodies by Direct ELISA

The half maximal effective concentration (EC₅₀) of the human-derivedTDP-43-specific antibodies for human TDP-43 and their targettarget-specificity was determined substantially as described in Example2 above. Briefly, 96 well microplates (Corning) were coated withrecombinant full length TDP-43 (Biogen Idec, USA), Escherichia coliextract and BSA (Sigma, Buchs, Switzerland), diluted to a concentrationof 5 μg/ml in carbonate ELISA coating buffer (pH 9.6) overnight at 4° C.Alternatively, 96 well half area microplates (Corning) were coated witha synthetic peptide covering residues 390 to 414 of the C-terminaldomain of TDP-43 with phosphorylation modification at residues 409/410(Schafer-N, DK) and BSA (Sigma, Buchs, Switzerland), diluted to aconcentration of 3.3 μg/ml in carbonate ELISA coating buffer (pH 9.6)overnight at 4° C. Non-specific binding sites were blocked for 1 hr. atroom temperature with PBS containing 2% BSA (Sigma, Buchs, Switzerland).Human TDP-43-specific antibodies were diluted to the indicatedconcentrations and incubated 1 hr. at room temperature. Binding wasdetermined using a donkey anti-human IgGγ-specific secondary antibodyconjugated with HRP (Jackson ImmunoResearch, USA), followed bymeasurement of HRP activity in a standard colorimetric assay. EC₅₀values were estimated by a non-linear regression using GraphPad Prismsoftware (San Diego, USA). Exemplary titration curves obtained with the41D1, 21G1, 31D2, and 8F8 antibodies are shown in FIG. 4.

As disclosed in Table 6, antibodies NI-205.41D1 (FIGS. 4A and E),NI-205.51C1 and NI.205-21G2 bound to human TDP-43 with high affinity atsubnanomolar EC₅₀ of 60 pM, 180 pM and 240 pM, respectively. No bindingwas observed to the phospho-TDP-43 C-terminal peptide. AntibodiesNI-205.1A9, NI-205.3F10, NI-205.14W3, NI-205.98H6, NI-205.44B2,NI-205.9E12A, NI-205.8A2, NI-205.15F12, NI-205.10D3, NI-205.38H2,NI-205.29E11, NI-205.9E12D, NI-205.31C11, NI-205.113C4, NI-205.25.25F3,NI-205.10H7, NI-205.8C10, and NI-205.87E7 bound to human TDP-43 but notto the phospho-TDP-43 C-terminal peptide with nanomolar EC₅₀. For theseantibodies EC₅₀ values ranged from 1 to 18 nM (see Table 6). AntibodyNI-205.21G1 (FIGS. 4B and F) bound to full length TDP-43 at 4.1 nM EC₅₀and recognized phospho-TDP-43 C-terminal peptide with lower affinity at49.5 nM EC₅₀. Antibodies NI-205.31D2 (FIGS. 4C and G), NI-205.14H5,NI-205.36D5, NI-205.19G5 and NI-205.68G5 showed preferential binding tothe phospho-TDP-43 C-terminal peptide with EC₅₀ values that ranged from0.7 to 17 nM (see Table 6). In contrast, antibodies NI-205.8F8,NI-205.8F8 (FIGS. 4D and H) and NI-205.20A1 bound exclusively to thehuman phospho-TDP-43 C-terminal peptide with high affinity at nanomolarEC₅₀ of 5 nM, 7 nM and 16 nM, respectively (see Table 6) consistent withthe idea that phosphorylation of serine 409 and/or serine 410 wasrequired for binding.

TABLE 6 EC₅₀ binding of human-derived TDP-43 antibodies to recombinanthuman TDP-43 and phospho TDP-43 C-terminal peptide. EC₅₀ [nM] Antibodyfull length TDP-43 phospho-TDP-43 peptide NI-205.41D1 0.06 no bindingNI-205.51C1 0.18 no binding NI-205.21G2 0.24 no binding NI-205.1A9 1.0no binding NI-205.3F10 1.4 no binding NI-205.14W3 1.5 no bindingNI-205.98H6 2.6 no binding NI-205.44B2 2.8 no binding NI-205.9E12A 3.7no binding NI-205.8A2 7.2 no binding NI-205.15F12 7.2 no bindingNI-205.10D3 7.3 no binding NI-205.38H2 8.2 no binding NI-205.29E11 10.4no binding NI-205.9E12D 11.2 no binding NI-205.31C11 11.0 no bindingNI-205.113C4 13.2 no binding NI-205.25F3 13.3 no binding NI-205.10H715.3 no binding NI-205.8C10 15.6 no binding NI-205.87E7 17.2 no bindingNI-205.21G1 4.1 49.5 NI-205.31D2 >41.0 0.69 NI-205.14H5 >100 0.90NI-205.36D5 >72.0 4.3 NI-205.19G5 >200 13.6 NI-205.68G5 >100 16.9NI-205.8F8 no binding 5.1 NI-205.58E11 no binding 6.9 NI-205.20A1 nobinding 15.8

Example 7: Human TDP-43 Antibody Binding Analysis by ELISA and WesternBlotting

Direct ELISAs

Direct ELISA assays were performed substantially as described inExample 1. Briefly, fragments of TDP-43 (SEQ ID NO:94) comprising aminoacids 2-106 (domain I), 99-204 (domain II), 183-273 (domain III),258-414 (domain IV) and full length TDP-43 (2-414) were coated ontoELISA plates at equal coating concentration of 6.6 μg/ml. Binding of thehuman-derived antibodies was determined by direct ELISA. Examples of theobtained results are shown in FIG. 5. Antibodies NI-205.41D1 (FIG. 5A),NI-205.14W3 (FIG. 5C), NI-205.44B2 (FIG. 5E), NI-205.10D3 (FIG. 5G), andNI-205.10H7 (FIG. 5L) bound specifically to TDP-43 domain IV (aa258-414). Antibody NI- NI-205.98H6 (FIG. 5D) bound specifically toTDP-43 domain III (aa 183-273). Antibodies NI-205.1A9 (FIG. 5B),NI-205.38H2 (FIG. 5H), and NI-205.31C11 (FIG. 5K) specificallyrecognized TDP-43 domain II (aa 99-204) whereas antibodies NI-205.9E12A(FIG. 5F), NI-205.29E11 (FIG. 50, NI-205.9E12D (FIG. 5J), andNI-205.8C10 (FIG. 5M) bound to TDP-43 domain I (aa 2-106). All thehuman-derived TDP-43 specific antibodies also recognized full lengthhuman TDP-43. To control for coating efficiency of the differentrecombinant TDP-43 domains, commercially available antibodies binding tofull length TDP-43 and a specific TDP-43 domain were used (FIG. 5W): i)Ab50930 (Abcam, UK), TDP-43 domain I; ii) TARDBP monoclonal antibody(M01), clone 2E2-D3 (Abnova 23435, Abnova, Taiwan), TDP-43 domain IIIand iii) Ab82695 (Abcam, UK), TDP-43 domain IV. Control antibodies boundto full length TDP-43 and their specific TDP-43 domain.

Binding to Distinct TDP-43 Domains by Western Blot

Recombinant full length TDP-43, TDP-43 domain I (amino acid residues2-106 of SEQ ID NO:94), TDP-43 domain II (amino acid residues 99-204 ofSEQ ID NO:94), TDP-43 domain III (amino acid residues 183-273 of SEQ IDNO:94), and TDP-43 domain IV (amino acid residues 258-414 of SEQ IDNO:94) were resolved by SDS-PAGE. Binding to specific TDP-43 domains ofthe human-derived antibodies was determined by Western Blot analysis.

Examples of the obtained results are shown in FIG. 6. AntibodiesNI-205.41D1 (FIG. 6A), NI-205.14W3 (FIG. 6F), NI-205.8A2 (FIG. 6H),NI-205.15F12 (FIG. 6I), NI-205.10D3 (FIG. 6J), NI-205.10H7 (FIG. 6L) andNI-205.21G1 (FIG. 6M) bound specifically to TDP-43 domain IV (aa258-414). Antibodies NI-205.51C1 (FIG. 6B), NI-205.3F10 (FIG. 6E) andNI-205.98H6 bound specifically to TDP-43 domain III (aa 183-273) whereasantibodies NI-205.21G2 (FIG. 6C), NI-205.1A9 (FIG. 6D) and NI-205.31C11(FIG. 6K) specifically recognized TDP-43 domain II (99-204 aa). Thesethirteen human-derived TDP-43 specific antibodies also recognized fulllength human TDP-43. Only antibody NI-205.10D3 (FIG. 6J) recognized anadditional unspecific signal, most probably an E. coli-derived proteincontaminant. NI-205.68G5 (FIG. 6N) and NI-205.20A1 (FIG. 6O), twoantibodies showing preferential or exclusive binding to thephospho-TDP-43 C-terminal peptide, did not recognize recombinant fulllength TDP-43 or any of its fragments. Commercially available TDP-43specific antibody 2E2-D3 (Abnova 23435, Abnova, Taiwan) (FIG. 6P) wasused as positive control for human TDP-43 detection whereas theanti-human IgG Fcγ-specific antibody (FIG. 6Q) was used as a negativecontrol.

Human-derived antibodies NI-205.44B2, NI-205.9E12A, NI-205.38H2,NI-205.29E11, NI-205.9E12D, NI-205.113C4, NI-205.25F3, NI-205.8C10 andNI-205.87E7 recognized full length TDP-43 but did not identify aspecific TDP-43 fragment (data not shown).

Example 8: Epitope Mapping with Synthetic Peptides (PepSpotting)

The epitopes recognized by the human-derived TDP-43 specific antibodieswithin the human TDP-43 protein were mapped using pepscan membranes(PepSpots, JPT Peptide Technologies, Berlin, Germany) with 101 linear15-meric peptides with 11 aa overlap between individual peptidescovering the entire human TDP-43 protein sequence. The pepscan mappingwas performed substantially as described in Example 3. Briefly, thepeptides were spotted onto nitrocellulose membranes that were thenactivated for 5 min. in methanol and subsequently washed at roomtemperature in TBS for 10 min. Table 7 summarizes binding epitopes forthe different human-derived TDP-43-specific antibodies identified usingPepSpot.

TABLE 7Binding epitopes within the human TDP-43 protein sequence for thedifferent human-derived TDP-43-specific antibodies identified usingPepSpot. NA-not applicable. Effect of phosphorylation at AntibodyBinding epitope Ser409 and/or Ser410 residues NI-205.41D1317-SINPAMMAAAQAAL-343 NA QSSWGMMGMLASQ- (SEQ ID NO: 323) NI-205.51C1201-DMTEDELREFF-211 NA (SEQ ID NO: 125) NI-205.21G2 121-KEYFSTF-127  NA(SEQ ID NO: 128) NI-205.3F10 213-QYGDVMDVFIP-223 NA (SEQ ID NO: 123)NI-205.98H6 249-IIKGISV-255  NA (SEQ ID NO: 315) NI-205.44B2345-NQSGPSG-351  NA (SEQ ID NO: 316) NI-205.8A2 381-AAIGWGSASNA-391 NA(SEQ ID NO: 124) NI-205.15F12 397-FNGGFGS-403  NA (SEQ ID NO: 317)NI-205.10D3 289-FGNSRGGGAGL-299 NA (SEQ ID NO: 318) 389-SNAGSGSGFNG-399(SEQ ID NO: 319) NI-205.113C4 133-VQVKKDL-139 NA (SEQ ID NO: 127)NI-205.10H7 269-QLERSGRFGGN-279 NA (SEQ ID NO: 320) NI-205.8C1017-EIPSEDD-23  NA (SEQ ID NO: 321) NI-205.87E7 9-EDENDEP-15  NA(SEQ ID NO: 126) NI-205.21G1 390-414 Ser409/Ser410 phosphorylationpartially inhibited binding NI-205.31D2 390-414 Bound to peptidephosphorylated at Ser409 and Ser410 NI-205.14H5 390-414 Bound to peptidephosphorylated at Ser409 and/or Ser410; weak bindingalso observed independent of phosphorylation NI-205.36D5 390-414Bound to peptide phosphorylated at Ser409; simultaneous Ser410phosphorylation abrogates binding NI-205.19G5 390-414 Bound to peptidephosphorylated at Ser409; simultaneous Ser410 phosphorylation abrogatesbinding NI-205.68G5 390-414 Bound to peptide phosphorylated at Ser409and/or Ser410 NI-205.8F8 390-414 not determined NI-205.58E11 390-414not determined NI-205.20A1 390-414 Bound to peptidephosphorylated at Ser409 and Ser410Determination of NI-205.41D1 Binding Epitope by ELISA Assays.

Full length TDP-43 (2-414) and TDP-43 C terminal fragments comprisingamino acids 258-414 (domain IV), 258-384, 258-375, 258-362, 258-353,258-319, 317-414 and 340-414 were coated onto ELISA plates at equalcoating concentration of 10 μg/ml. Binding of NI-205.41D1 antibody tospecific TDP-43 fragments was determined by direct ELISA. Examples ofthe data obtained are provided in FIG. 7A. NI-205.41D1 bound to allrecombinant fragments except fragments 258-319 and 340-414, indicatingthat NI-205.41D1 binding epitope was in the C-terminal TDP-43 region317-353. NI-205.41D1 antibody bound to full length TDP-43.

Full length TDP-43 (2-414), wild-type TDP-43 domain IV comprisingresidues 258-414 of SEQ ID NO:94 (TDP-43 258-414) and a mutant TDP-43domain IV (TDP-43 258-414 AMM321GGG) carrying the A to G substitution atresidue 321, M to G substitution at residue 322, and M to G substitutionat residue 323 were coated onto ELISA plates at equal coatingconcentration of 10 μg/ml. Binding of NI-205.41D1 antibody to specificTDP-43 domain IV variants was determined by direct ELISA (FIG. 7B).NI-205.41D1 specifically bound to full length TDP-43 and to wild typeTDP-43 domain IV, but not to the mutant TDP-43 domain IV, indicatingthat one or more of the mutated residues was essential for NI-205.41D1binding to human TDP-43. To control for coating efficiency of thedifferent recombinant TDP-43 species, commercially available antibody12892-1-AP binding to full length TDP-43 was used.

Synthetic biotinylated peptides comprising residues 316-353 (TDP-43316-353), 316-343 (TDP-43 316-343) and 316-333 (TDP-43 316-333) of SEQID NO:94 were coated on streptavidin-coated plates at equal coatingconcentration of 10 μg/ml. Binding of NI-205.41D1 antibody to specificTDP-43 C-terminal peptides was determined by direct ELISA (FIG. 7C).NI-205.41D1 specifically bound to peptides TDP-43 316-353 and TDP-43316-343 but not to peptide TDP-43 316-333. This result is consistentwith the idea that residues 334-343 of SEQ ID NO:94 at the TDP-43C-terminal region are involved in NI-205.41D1 antibody binding to humanTDP-43. Our results are consistent with an understanding that thebinding epitope of antibody NI-205.41 is discontinuous between residues317-343 of SEQ ID NO:94 and is formed by two independent bindingregions: the first one comprising residues 321-323 of SEQ ID NO:94 andthe second one comprising residues 334-343 of SEQ ID NO:94.

Example 9: The Human-Derived TDP-43 Specific Antibodies Interact withNative TDP-43

Pure full-length TDP-43 protein has a natural propensity to aggregate.Therefore, only very small quantities of soluble, full-length TDP-43were recovered under standard purification conditions. We thus developeda recombinant expression and purification strategy for isolating largequantities of functional 6×His-SUMO-tagged full-length human TDP-43 fromEscherichia coli using KSCN and arginine, mild chaotropic agents knownto preserve native protein structure while preventing proteinaggregation.

Plasmids:

Polynucleotides encoding human full-length TDP-43 (1-414 of SEQ IDNO:94) and its truncations residues 101-265 and residues 220-414 of SEQID NO:94 were amplified using standard procedures and subcloned into amodified pET19-b (Novagen) vector resulting in 6×His and SUMO tags atthe N-terminus of the protein encoded by the amplified polynucleotides.A schematic representation of the 6×His/SUMO tagged recombinantpolypeptides is shown in FIG. 8B.

Protein expression and purification: 6×His/SUMO tagged TDP-43 expressionplasmids were transformed into BL21 STAR™ (DE3) Escherichia coli(INVITROGEN™). Bacterial cultures were grown to an OD600 of 1.0 at 37°C. and induced with 1 mM IPTG for 16 h at 18° C. After pelleting, cellswere lysed by microfluidization in purification buffer with proteaseinhibitors (40 mM HEPES (pH 7.5), 1.5 M KSCN, and 1 mMtris(2-carboxyethyl)phosphine (TCEP), 1 mM PMSF, 5 μM pepstatin, 1 mMbenzamine, 10 μM bestatin, 10 μM E-64, 20 μM leupeptin, 1.5 μMaprotinin). To generate properly folded full-length TDP-43, TDP-43(101-265) and TDP-43 (220-414), the corresponding 6×His-SUMO taggedfusions proteins were purified on Ni-NTA agarose resin (QIAGEN®)following the manufacturer's instructions, using purification buffer forbinding and washing. Bound proteins were eluted from Ni-NTA resin withthe same buffer containing 250 mM imidazole and further purified on apreparative S200 size exclusion column (GE Healthcare) following themanufacturer's instructions using purification buffer. Fractionscontaining monomeric TDP-43 proteins were pooled, and re-formulated in abuffer containing 40 mM HEPES (pH 7.5), 400 mM arginine, 1 mM TCEP bydialysis. 6×His-SUMO-TDP43 (101-265) was additionally purified using thesame purification strategy but altering buffer compositions bysubstituting 1.5 M KSCN with 0.5M KCl. To prepare unfolded 6×His-TDP-43,cells were lysed by microfluidization in a Tris-imidazole buffer (50 mMTris (pH 7.5), 20 mM imidazole, 150 mM NaCl) with protease inhibitors.The insoluble pellet was washed sequentially with the following bufferscontaining protease inhibitors: B-PER buffer (Pierce) containing 2 mMMgCl₂, followed by Tris-imidazole buffer. Washed pellets were nextsolubilized in 8 M urea, 20 mM sodium phosphate (pH 7.8). Urea solublematerial was then purified on Ni-NTA agarose resin following themanufacturer's instructions, using denaturing washing and elutionconditions. Sedimentation and diffusion coefficients determination (FIG.8C) as well as SDS-PAGE separation (FIG. 8A) were carried out bystandard methods. (Laue, T. et al.. (1992) in AnalyticalUltracentrifugation in Biochemistry and Polymer Science (Harding, S. E.,ed) Royal Society of Chemistry, Cambridge, UK).

Capture ELISA: 96 well plates (Thermo Fisher Scientific) were coatedwith anti 6×His mouse monoclonal antibody (Clontech) diluted to aconcentration of 1 μg/ml in PBS buffer (137 mM NaCl, 8.05 mM Na2HPO4,1.5 mM KH2PO4, 2.7 mM KCl, pH 7.4) at 4° C. overnight. Non-specificbinding sites were blocked for 1 hr at RT with PBS containing 1% BSA(Sigma) and 0.05% TWEEN®20 (Fisher Scientific). 6×His-SUMO-TDP43proteins at a concentration of 1.7 μM were allowed to bind toantibody-coated plates for 1 hr at RT in PBS buffer containing 1% BSA,300 mM arginine and 0.1% PEG 5000, pH 7.5. Plates were incubated for 1hr at RT with human antibodies of present invention, titrated in athree-fold dilution series, followed by HRP conjugated anti-human IgG Fcγ (Jackson ImmunoResearch) in PBST containing 1% BSA. HRP activity wasmeasured in a standard colorimetric assay. EC50 values were calculatedusing a four-parameter logistic curve fit on Softmax pro software(Molecular Devices).

RNA Binding Assay:

Equilibrium binding affinities of TDP-43 constructs for RNA weredetermined using fluorescence polarization. 5′TYETM fluorophore labeledRNA substrates, specific RNA (TYETM-UGUGUGUGUGUG) (SEQ ID NO: 312) andRNA control (TYETM-UUUUUUUUUUUU) (SEQ ID NO: 313) (Integrated DNATechnologies), were incubated at a concentration of 5 nM for 30 min at25° C. with TDP-43 (final concentrations 0 to 20 μM) in incubationbuffer as indicated. Fluorescence polarization was measured with theplate reader fluorometer Envision (PerkinElmer) at each concentration ofTDP-43 in a 96 well plate format with an excitation at 645 nm andemission at 665. K_(dS) were calculated using the quadratic equation fortight binding (Morrison equation) using Sigmaplot (Systat SoftwareInc.). To determine the stoichiometry of RNA/TDP-43 binding, the samefluorescence polarization experimental set up was used with the additionof 95 nM of unlabeled RNA of the same sequence for a total 100 nM RNAconcentration (>7× higher than the previously determined K_(d) values).Stoichiometries were calculated by measuring the intercept of twostraight lines, one fit using data points of the partially bound state,and the other using data points of the fully bound state.

The recombinant full-length TDP-43 was monomeric by sedimentationanalysis (FIG. 8C) in buffer containing 400 mM arginine. A minimum of300 mM arginine was maintained in analytical assays when monomericnative TDP-43 was desired, because aggregation was observed at lowerarginine concentrations. Consistent with arginine not adverselyaffecting TDP-43 activity, our recombinant, full-length TDP-43 binds anRNA of previously established specific sequence (UGUGUGUGUGUG (SEQ IDNO:312)) with at least 30 fold higher affinity than to a genericunspecific sequence (UUUUUUUUUUUU (SEQ ID NO:313)) (FIG. 9).

We also purified a 6×His/SUMO tagged fragment of TDP-43 comprising aminoacid residues 101-265 of SEQ ID NO: 94 from which the C-terminal domain(amino acids 265 to 414) known to mediate aggregation was removed. The6×His/SUMO tagged 101-265 truncated TDP-43 fragment, purified with orwithout chaotropes maintained a binding affinity to the specific RNAsequence very similar to the previously reported K_(D) (˜14 nM) in spiteof the very different analytical methods used (FIG. 9). See, Kou NucleicAcids Res. 2009, 37:1799-808. The stoichiometry of RNA/TDP-43 bindingwas also the same regardless of purification strategy, indicating thatthere was no significant difference in the denatured protein populationbetween the two preparations (FIG. 9). Together, these data indicatedthat the purified recombinant pure full length TDP-43 was in its nativefolding state.

We measured the binding affinity of the human-derived TDP-43 specificantibodies provided herein to properly folded 6×His/SUMO tagged TDP-43using a capture ELISA in which native TDP-43 was immobilized withoutdirect adsorption on the surface of the ELISA plate. This was achievedby immobilizing an anti 6×His antibody which then captured nativeTDP-43. Binding to the anti 6×His tag by folded 6×His/SUMO taggedfull-length human TDP-43 was achieved at 300 mM arginine concentration,ensuring a monomeric state of TDP-43. After this immobilization step,binding by human antibodies was tested in regular buffers withoutinterference of TDP-43 aggregation. An example of the titration curvesgenerated is shown in FIG. 10. Table 8 summarizes affinities (EC₅₀ [nM])to folded, full length 6×His/SUMO tagged TDP-43, or to a 6×His/SUMOtagged truncation construct containing the C-terminal, aggregation proneregion of TDP-43 (residues 220-414 of SEQ ID NO: 94) by this captureELISA method.

TABLE 8 EC₅₀ [nM] binding of human-derived TDP-43 antibodies to properlyfolded recombinant 6xHis/SUMO tagged full length TDP-43 and 6xHis/SUMOtagged truncated TDP-43 comprising residues 220-414. Antibody fulllength TDP-43 TDP-43 residues 220-414 NI-205.41D1 0.07 0.10 NI-205.51C11.1 no binding NI-205.21G2 3.6 no binding NI-205.1A9 >98 no bindingNI-205.3F10 >90 no binding NI-205.14W3 7.4 1.6 NI-205.98H6 46 20NI-205.44B2 >125 33.5 NI-205.9E12A no binding — NI-205.8A2 34 4.6NI-205.15F12 16.3 9.7 NI-205.10D3 >90 20.5 NI-205.38H2 >120 no bindingNI-205.29E11 >100 >97 NI-205.9E12D no binding no binding NI-205.31C1121.1 no binding NI-205.113C4 no binding no binding NI-205.25F3 >120 >100NI-205.10H7 30.9 6.45 NI-205.8C10 no binding — NI-205.87E7 no binding nobinding NI-205.21G1 4.4 0.33 NI-205.31D2 41.0 19.4 NI-205.14H5 >100 —NI-205.36D5 >72.0 >60 NI-205.19G5 no binding — NI-205.68G5 no binding —NI-205.8F8 >94 — NI-205.58E11 no binding — NI-205.20A1 no binding —

Example 10: Assessment of Human-Derived TDP-43 Antibody Binding toTDP-43 in FTLD-U Case and Control Hippocampal Tissues

Human cortical, hippocampal, and spinal cord FTLD-U and control tissueswere obtained from the IDIBAPS Biobank (Banc de Teixits Neurologics,Barcelona). Immunohistochemistry was performed on 5 μm thick paraffinembedded sections using EDTA-based epitope retrieval prior to conductingthe otherwise standard immunohistochemical procedures with Elite ABCkits (Vector Laboratories) with DAB (Thermo Scientific).Immunohistochemistry was performed using the human TDP-43 antibodies ofthe invention at 50 nM concentration. Control stainings were done usingmouse monoclonal antibody 2E2 against human TDP-43 (Abnova), rabbitpolyclonal antibody p409/p410 raised against TDP-43 p409/p410(CosmoBio), and rabbit polyclonal antibody p409/p410 raised againstTDP-43 p409/p410 (CosmoBio).

The capacity of the human derived anti-TDP-43 antibodies describedherein to recognize native and pathological forms of TDP-43 wascharacterized by immunohistochemistry experiments on human FTLD-U case(10) and control (7) hippocampal tissues. TDP-43 is a predominantlynuclear protein which shuttles to and from the cytoplasm. Underpathological conditions it accumulates in the nucleus and particularlyin the cytoplasm, and the pathology is typicallycharacterized/classified based on cellular localization; NCI—neuronalcytoplasmic inclusion, NII—neuronal intranuclear inclusion, anddystrophic neuritic pathology (review Mackenzie et al., LancetNeurology, 9: 995-1007 (2010)). In FTLD-U and ALS patient tissues, theprotein is also found to be phosphorylated. The human TDP-43 bindingcharacteristics of the antibodies disclosed herein were compared to thatof commercially available antibodies commonly used to diagnose casespost-mortem. The 2E2-D3 control antibody (epitope mapped to aa205-222;Zhang et al., Neurosci. Lett., 434:170-174 (2008)) recognized nuclearand cytoplasmic TDP-43 accumulation in hippocampal pyramidal neurons(FIG. 11A) and granule cells (FIG. 11B), as well as TDP-43 in dystrophicneurites (FIG. 11C). On control case tissues, 2E2-D3 recognizedpredominantly nuclear TDP-43. The phosphorylation specific antibody,p403/p404 (raised against CNGGFGS(p)S(p)MDSK (SEQ ID NO:324); Hasegawaet al., Ann Neurol, 64(1):60-70 (2008)) recognized (FIG. 11D)cytoplasmic TDP-43 in pyramidal cells, (FIG. 11E) nuclear andcytoplasmic accumulation in granule cells, as well as (FIG. 11F) TDP-43in dystrophic neurites. A second phosphorylation specific antibody,p409/p410 (raised against CMDSKS(p)S(p)GWGM (SEQ ID NO:325); Hasegawa etal., Ann Neurol, 64(1):60-70 (2008)) bound to TDP-43 accumulating in thenucleus and cytoplasm of (FIG. 11G) pyramidal cells and (FIG. 11H)granule cells, as well as (FIG. 11I) TDP-43 in dystrophic neurites.

The human-derived anti-TDP-43 antibodies described herein displayedvarious staining patterns, including binding to nuclear, cytoplasmic,and neuritic forms of TDP-43. Several of the human-derived anti-TDP-43antibodies described herein specifically bound to disease forms ofTDP-43, compared to staining on control case tissues from healthyindividuals. (FIG. 11, and Table 9). For example, antibodiesNI-205.68G5, NI-205.14W3, NI-205.21G1, and NI-205.41D1, selectivelybound to pathological forms, i.e. to neuritic TDP-43, and nuclear andcytoplasmic TDP-43 in hippocampal granule cells. Antibodies NI-205.14W3,NI-205.21G1, and NI-205.41D1 specifically bound to pathological forms ofTDP-43 in FTLD-U patient tissues without binding to control case tissues(compare for NI-205.41D1 staining in FTLD-U patient tissues (FIG. 11Y)and control case tissue (FIG. 11Z)).

Antibody (FIG. 11J) NI-205.10D3 bound predominantly to nuclear TDP-43,while (FIG. 11K) NI-205.8C10 bound to TDP-43 in cytoplasm and axons.Unlike the control anti-TDP-43 antibodies analyzed, a sub-set of thehuman anti-TDP-43 antibodies reported herein bound predominantlycytoplasmic TDP-43, rather than nuclear TDP-43. Antibodies that boundpredominantly cytoplasmic TDP-43 include (FIG. 11L) NI-205.15F12, (FIG.11M) NI-205.8A2, (FIG. 11N) NI-205.3F10, (FIG. 11O) NI-205.21G2, (FIG.11P) NI-205.8F8, (FIG. 11Q) NI-205.31C11, (FIG. 11R) NI-205.36D5, (FIG.11S) NI-205.31D2, (FIG. 11T) NI-205.10H7, and (FIG. 11U) NI-205.14H5.Antibodies (FIG. 11V) NI-205.68G5, (FIG. 11W) NI-205.14W3, (FIG. 11X)NI-205.21G1, and (FIG. 11Y) NI-205.41D1 bound to neuritic TDP-43 andTDP-43 accumulating in the nucleus and cytoplasm in hippocampal granulecells.

TABLE 9 Assessment of human-derived TDP-43 antibody binding to TDP-43 inFTLD-U case and control hippocampal tissues. Case and control sampleswere also stained with 2E2-D3, p403/p404, p409/p410 control anti-TDP-43antibodies. ND - not determined Antibody Staining byImmunohistochemistry Antibody FTLD-U Case tissue Control tissueNI-205.41D1 cytoplasmic and nuclear in no binding granule cells +neuritic NI-205.51C1 no binding ND NI-205.21G2 cytoplasmic ND NI-205.1A9no binding ND NI-205.3F10 cytoplasmic cytoplasmic NI-205.14W3cytoplasmic and no binding, cytoplasmic in nuclear in granule neurons(in some cases) cells + neuritic NI-205.98H6 no binding ND NI-205.44B2no binding ND NI-205.9E12A no binding ND NI-205.8A2 cytoplasmiccytoplasmic NI-205.15F12 cytoplasmic in granule cells ND NI-205.10D3nuclear nuclear NI-205.38H2 no binding ND NI-205.29E11 no binding NDNI-205.9E12D no binding ND NI-205.31C11 cytoplasmic cytoplasmicNI-205.113C4 no binding ND NI-205.25F3 no binding ND NI-205.10H7cytoplasmic cytoplasmic NI-205.8C10 cytoplasmic + axonal (one no bindingcase) NI-205.87E7 no binding ND NI-205.21G1 cytoplasmic and no bindingand nuclear in granule cells + neuritic NI-205.31D2 cytoplasmiccytoplasmic NI-205.14H5 cytoplasmic cytoplasmic NI-205.36D5 cytoplasmiccytoplasmic NI-205.19G5 no binding ND NI-205.68G5 cytoplasmic + neuritic(one ND case) NI-205.8F8 cytoplasmic cytoplasmic NI-205.58E11 no bindingND NI-205.20A1 no binding ND 2E2-D3 Nuclear, cytoplasmic and Nuclear(cytoplasmic in neuritic some controls) p403/p404 Nuclear, cytoplasmicand no binding neuritic p409/p410 Nuclear, cytoplasmic and No bindingneuritic

The screen for human anti-TDP-43 antibodies using denatured recombinantTDP-43 and TDP-43 390-414 peptide phosphorylated at residues 5409 and5410 resulted in the generation of antibodies that cover most naturaland disease-related epitopes of human TDP-43. The human anti-TDP-43antibodies disclosed herein identify new and interesting epitopes ofTDP-43, and provide novel conformational information specific to thedisease process of TDP-43 proteinpathies. For example, the NI-205.14W3,NI-205.21G1, and NI-205.41D1 bound to TDP-43 with high affinity and werespecific to pathological forms of TDP-43 on FTLD-U tissues in comparisonto control cases. While these three antibodies had similar pathologicalTDP-43 specific staining pattern in immunohistochemistry, theyrecognized distinct epitopes in the aggregation prone C-terminal regionof TDP-43: NI-41D1 bound to a discontinuous epitope in the C-terminalportion of TDP-43 and NI-21G1 bound to a phosphorylation prone region ofTDP-43. Additionally, the NI-205.14H5 and NI-205.31D2 antibodies hadhigh affinity for TDP-43 phosphorylated at one or both of residues 5409and 5410, and specifically stained cytoplasmic TDP-43 inimmunohistochemistry. Furthermore, NI-205.21G2 and NI-205.51C1 alsodemonstrated high affinity for TDP-43, and bound to epitopes N-terminalto the predicted caspase cleavage site, with NI-205.51C1 binding toRNA-recognition motif 2 (RRM2). NI-205.10D3 specifically stained nuclearTDP-43 in both FTLD-U and control case tissues, suggesting that it boundto endogenous/native forms of TDP-43.

Example 11: Acute Brain Penetration Study

TDP-43_G348C transgenic mice (Swamp et al., Brain 134 (2011), 2610-2626)are intraperitoneally injected with 30 mg/kg human anti-TDP-43 antibodyor equal volume of PBS at day 1 and day 4. At day 5, mice are perfusedunder anesthesia with PBS containing 1 Unit/ml heparin. Blood, brain andspinal cord are collected for analyses. Right hemisphere of the brain isfrozen at −80° C., left hemisphere of the brain and the spinal cord arepost fixed in 10% neutralized formalin at 4° C. for two days beforebeing embedded in paraffin block and sectioned. Plasma is stored at −80°C. in aliquots.

Brain protein extraction: brain protein fractions are extracted usingstandard experimental methods, for example, frozen right hemisphere isweighed and homogenized in 5 volumes (5 mL/g of wet tissue) of asolution containing 50 mM NaCl, 0.2% diethylamine, protease inhibitors(Roche Diagnostics GmbH) and phosphatase inhibitor (Roche DiagnosticsGmbH). Samples are then transferred to polycarbonate tubes and addedanother 5 volume of homogenization solution, and kept on ice for 30 min.Soluble fraction is then collected after centrifugation at 100,000 g, 4°C. for 30 min. This soluble fraction is used in human IgG assay. Thepellet is resuspended in 3 volumes of PBS with protease and phosphataseinhibitor. After centrifugation at 16,000 g, 4° C. for 30 min,supernatants and pellets are stored separately at −80° C. for furtherinsoluble TDP-43 extraction.

Human antibody and TDP-43 is detected and quantitated in the brainprotein extracts using standard experimental methods. For example, humanIgG-specific sandwich ELISA: 2 μg/ml of goat anti-human IgG Fab(Jackson) in 50 mM carbonate ELISA coating buffer (pH9.6) is used ascapture antibody. Half-area 96-well microtitre plates are coated with 30μl/well with capture antibody at 4° C. over night. The plate is thenwashed 4 times with PBS containing 0.1% TWEEN®20 before incubating with50 μl/well PBS containing 2% BSA at room temperature for one hour.Soluble fractions of brain extracts, plasma samples and human antibodystandard are diluted in PBS containing 2% BSA and 0.1% TWEEN® 20. 30 μlof the diluted samples are added into each well and incubated at roomtemperature for one hour. The plate is then washed with 200 μl/well PBScontaining 0.1% TWEEN®20 for four times before incubated withHRP-conjugated donkey anti-human Fcγ (Jackson, diluted at 1:10,000 inPBS containing 2% BSA and 0.1% TWEEN®20) at room temperature for onehour. The plate is then washed with 200 μl/well PBS containing 0.1%TWEEN®20 for four times before adding 20 μl/well TMB (1:20 in 10 mMcitrate solution pH=4.1). The reaction is then stopped by adding 10 μl1M H₂SO₄ to each well. Antibody standard curve is obtained from serialdilutions of control antibody. Antibody concentrations in plasma andbrain samples are calculated according to the standards. Brain human IgGlevel is then converted to μg antibody/gram fresh brain tissue.

Neuronal penetration of the administered human anti-TDP-43 antibody isdetected by immunohistological staining of the brain tissue sectionsobtained from human anti-TDP-43 antibody treated and control animals.For example, free-floating tissue sections are washed in Tris TRITON®pH7.4 (50 mM Tris, 150 mM NaCl, 0.05% TRITON® X-100), incubated in 1%H₂O₂ PBS for 30 min, and incubated with a blocking solution containing2% normal goat- and horse serum in Tris TRITON® and with additional 0.2%TRITON® X-100 for 1 h at room temperature. The sections are thenincubated with biotinylated donkey anti-human IgG (H+L) (JacksonImmunoresearch Labs, 709-065-149) at 1:200 in blocking solution for 16 hat 4° C. with agitation at 100 rpm to detect neuronal human IgG. Thetissue-bound biotinylated antibody is visualized by peroxidasechromogenic reaction using the VECTASTAIN® Elite ABC kit (VectorLaboratories, PK6100, 1:100). The enzymatic reaction is stopped with icecold PBS and the sections is washed in PBS 3 times. The sections arethen mounted on glass slides and air dried over night before they arecounterstained with hemalum solution (Carl Roth GmbH+Co. T865.1). Afterdehydration steps, the slides are covered with coverslips before beingscanned with the Olympus dotSlide 2.1 virtual microscopy system.Neuronal anti-human IgG staining observed in the antibody treatedanimals, but not in the control animals, indicates that the humananti-TDP-43 antibody enters the neurons.

Example 12: Chronic Study with Anti-TDP-43 Antibodies

TDP-43_G348C transgenic mice (Swamp et al., Brain 134 (2011), 2610-2626)are intraperitoneally injected with 10 mg/kg, 3 mg/kg of antibodysolution, or equal volume of PBS control. Each treatment group has 20-25mice. The treatment is carried out once a week for 26 weeks.Alternatively, the treatment is carried out twice a week for 13 weeks.Body weight is monitored every two weeks. Mice are perfused underanesthesia at the end of the treatment period. Brain, spinal cord andblood is collected. Half brain and spinal cord are post-fixed in 10%formalin for three days before being embedded in paraffin block. 4-6 μmthick sections cut from these tissue blocks are used forimmunohistochemistry studies. The other half brain is weighted and deepfrozen at −80° C. for biochemical analyses.

Drug effects are evaluated by comparing the level and distribution ofTDP-43, including the level and distribution of pathological forms ofTDP-43 in antibody treated and control animals usingimmunohistochemistry. Tissue samples obtained from antibody treated andcontrol animals are stained with an anti-TDP-43 antibody, e.g., ananti-TDP-43 antibody specific for pathological forms of TDP-43, usingstandard histological methods. In one embodiment, the antibody used inthe histochemical analysis is the same as the antibody administered tothe animals. In another embodiment, the antibody used in thehistochemical analysis is different from the antibody administered tothe animals. Therapeutic efficacy of the human anti-TDP-43 antibodiesdisclosed herein is indicated by a reduction in the level, or absence ofpathological forms of TDP-43 in antibody treated animals relative tocontrol animals.

Drug effects are also evaluated by comparing the level of TDP-43,including the level of pathological forms of TDP-43 in antibody treatedand control animals using ELISA or Western-blot. Therapeutic efficacy ofthe human anti-TDP-43 antibodies disclosed herein is indicated by areduction in the level, or absence of pathological forms of TDP-43 inantibody treated animals relative to control animals.

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and any compositions or methodswhich are functionally equivalent are within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description and accompanying drawings.Such modifications are intended to fall within the scope of the appendedclaims.

What is claimed is:
 1. A polynucleotide linked to a heterologous nucleicacid, wherein the polynucleotide is selected from the group consistingof: (a) a polynucleotide encoding an immunoglobulin heavy chain or afragment thereof comprising a heavy chain variable region (VH)comprising complementarity determining regions (CDRs) 1, 2, and 3 withthe amino acid sequences set forth in SEQ ID NOs: 11, 12, and 13,respectively, and wherein the VH when paired with a light chain variableregion (VL) comprising the amino acid sequence set forth in SEQ ID NO:14binds to TAR DNA-binding protein of 43 kDa (TDP-43); (b) apolynucleotide encoding an immunoglobulin light chain or a fragmentthereof comprising a VL comprising CDRs 1, 2, and 3 with the amino acidsequences set forth in SEQ ID NOs: 15, 16, and 17, respectively, andwherein the VL when paired with a VH comprising the amino acid sequenceset forth in SEQ ID NO: 10 binds to TDP-43; (c) a polynucleotideencoding (i) an immunoglobulin heavy chain or a fragment thereofcomprising a VH comprising CDRs 1, 2, and 3 with the amino acidsequences set forth in SEQ ID NOs: 11, 12, and 13, respectively; and(ii) an immunoglobulin light chain or a fragment thereof comprising a VLcomprising CDRs 1, 2, and 3 with the amino acid sequences set forth inSEQ ID NOs: 15, 16, and 17, respectively; (d) a polynucleotide encodingan immunoglobulin heavy chain or a fragment thereof comprising a VHcomprising the amino acid sequence set forth in SEQ ID NO:10, whereinthe VH when paired with a VL comprising the amino acid sequence setforth in SEQ ID NO: 14 binds to TDP-43; (e) a polynucleotide encoding animmunoglobulin light chain or a fragment thereof comprising a VLcomprising the amino acid sequence set forth in SEQ ID NO:14, whereinthe VL when paired with a VH comprising the amino acid sequence setforth in SEQ ID NO:10 binds to TDP-43; (f) a polynucleotide encoding animmunoglobulin heavy chain or a fragment thereof comprising a VHcomprising the amino acid sequence set forth in SEQ ID NO:10 and animmunoglobulin light chain or a fragment thereof comprising a VLcomprising the amino acid sequence set forth in SEQ ID NO:14; (g) apolynucleotide encoding an immunoglobulin heavy chain or a fragmentthereof comprising a heavy chain variable region (VH) comprisingcomplementarity determining regions (CDRs) 1, 2, and 3 with the aminoacid sequences set forth in SEQ ID NOs: 131, 132, and 133, respectively,and wherein the VH when paired with a light chain variable region (VL)comprising the amino acid sequence set forth in SEQ ID NO:134 binds toTDP-43; (h) a polynucleotide encoding an immunoglobulin light chain or afragment thereof comprising a VL comprising CDRs 1, 2, and 3 with theamino acid sequences set forth in SEQ ID NOs: 135, 136, and 137,respectively, and wherein the VL when paired with a VH comprising theamino acid sequence set forth in SEQ ID NO: 130 binds to TDP-43; (i) apolynucleotide encoding (i) an immunoglobulin heavy chain or a fragmentthereof comprising a VH comprising CDRs 1, 2, and 3 with the amino acidsequences set forth in SEQ ID NOs: 131, 132, and 133, respectively; and(ii) an immunoglobulin light chain or a fragment thereof comprising a VLcomprising CDRs 1, 2, and 3 with the amino acid sequences set forth inSEQ ID NOs: 135, 136, and 137, respectively; (j) a polynucleotideencoding an immunoglobulin heavy chain or a fragment thereof comprisinga VH comprising the amino acid sequence set forth in SEQ ID NO:130,wherein the VH when paired with a VL comprising the amino acid sequenceset forth in SEQ ID NO: 134 binds to TDP-43; (k) a polynucleotideencoding an immunoglobulin light chain or a fragment thereof comprisinga VL comprising the amino acid sequence set forth in SEQ ID NO:134,wherein the VL when paired with a VH comprising the amino acid sequenceset forth in SEQ ID NO:130 binds to TDP-43; (l) a polynucleotideencoding an immunoglobulin heavy chain or a fragment thereof comprisinga VH comprising the amino acid sequence set forth in SEQ ID NO:130 andan immunoglobulin light chain or a fragment thereof comprising a VLcomprising the amino acid sequence set forth in SEQ ID NO:134; (m) apolynucleotide encoding an immunoglobulin heavy chain or a fragmentthereof comprising a heavy chain variable region (VH) comprisingcomplementarity determining regions (CDRs) 1, 2, and 3 with the aminoacid sequences set forth in SEQ ID NOs: 260, 261, and 262, respectively,and wherein the VH when paired with a light chain variable region (VL)comprising the amino acid sequence set forth in SEQ ID NO:263 binds toTDP-43; (n) a polynucleotide encoding an immunoglobulin light chain or afragment thereof comprising a VL comprising CDRs 1, 2, and 3 with theamino acid sequences set forth in SEQ ID NOs: 264, 265, and 266,respectively, and wherein the VL when paired with a VH comprising theamino acid sequence set forth in SEQ ID NO: 259 binds to TDP-43; (o) apolynucleotide encoding (i) an immunoglobulin heavy chain or a fragmentthereof comprising a VH comprising CDRs 1, 2, and 3 with the amino acidsequences set forth in SEQ ID NOs: 260, 261, and 262, respectively; and(ii) an immunoglobulin light chain or a fragment thereof comprising a VLcomprising CDRs 1, 2, and 3 with the amino acid sequences set forth inSEQ ID NOs: 264, 265, and 266, respectively; (p) a polynucleotideencoding an immunoglobulin heavy chain or a fragment thereof comprisinga VH comprising the amino acid sequence set forth in SEQ ID NO:259,wherein the VH when paired with a VL comprising the amino acid sequenceset forth in SEQ ID NO:263 binds to TDP-43; (q) a polynucleotideencoding an immunoglobulin light chain or a fragment thereof comprisinga VL comprising the amino acid sequence set forth in SEQ ID NO:263,wherein the VL when paired with a VH comprising the amino acid sequenceset forth in SEQ ID NO:259 binds to TDP-43; and (r) a polynucleotideencoding an immunoglobulin heavy chain or a fragment thereof comprisinga VH comprising the amino acid sequence set forth in SEQ ID NO:259 andan immunoglobulin light chain or a fragment thereof comprising a VLcomprising the amino acid sequence set forth in SEQ ID NO:263.
 2. Thepolynucleotide linked to a heterologous nucleic acid of claim 1, whereinthe heterologous nucleic acid is a regulatory element.
 3. Thepolynucleotide linked to a heterologous nucleic acid of claim 2, whereinthe regulatory element is a promoter, an enhancer, a ribosome bindingsite, or a transcription terminator.
 4. The polynucleotide linked to aheterologous nucleic acid of claim 1, wherein the heterologous nucleicacid encodes a secretory signal peptide.
 5. The polynucleotide linked toa heterologous nucleic acid of claim 4, wherein the secretory signalpeptide is a mammalian signal peptide.
 6. The polynucleotide linked to aheterologous nucleic acid of claim 5, wherein the mammalian signalpeptide is from tissue plasminogen activator.
 7. An isolated host cellcomprising the polynucleotide linked to a heterologous nucleic acid ofclaim
 1. 8. The isolated host cell of claim 7, wherein the host cell isa Chinese Hamster Ovary (CHO) cell.
 9. A cDNA comprising apolynucleotide encoding a polypeptide comprising: an immunoglobulinheavy chain or a fragment thereof comprising a heavy chain variableregion (VH) comprising complementarity determining regions (CDRs) 1, 2,and 3 with the amino acid sequences set forth in SEQ ID NOs:11, 12, and13, respectively, and wherein the VH when paired with a light chainvariable region (VL) comprising the amino acid sequence set forth in SEQID NO:14 binds to TDP-43; an immunoglobulin light chain or a fragmentthereof comprising a VL comprising CDRs 1, 2, and 3 with the amino acidsequences set forth in SEQ ID NOs:15, 16, and 17, respectively, andwherein the VL when paired with a VH comprising the amino acid sequenceset forth in SEQ ID NO:10 binds to TDP-43; an immunoglobulin heavy chainor a fragment thereof comprising a heavy chain variable region (VH)comprising complementarity determining regions (CDRs) 1, 2, and 3 withthe amino acid sequences set forth in SEQ ID NOs:131, 132, and 133,respectively, and wherein the VH when paired with a light chain variableregion (VL) comprising the amino acid sequence set forth in SEQ IDNO:134 binds to TDP-43; an immunoglobulin light chain or a fragmentthereof comprising a VL comprising CDRs 1, 2, and 3 with the amino acidsequences set forth in SEQ ID NOs:135, 136, and 137, respectively, andwherein the VL when paired with a VH comprising the amino acid sequenceset forth in SEQ ID NO:130 binds to TDP-43; an immunoglobulin heavychain or a fragment thereof comprising a heavy chain variable region(VH) comprising complementarity determining regions (CDRs) 1, 2, and 3with the amino acid sequences set forth in SEQ ID NOs:260, 261, and 262,respectively, and wherein the VH when paired with a light chain variableregion (VL) comprising the amino acid sequence set forth in SEQ IDNO:263 binds to TDP-43; or an immunoglobulin light chain or a fragmentthereof comprising a VL comprising CDRs 1, 2, and 3 with the amino acidsequences set forth in SEQ ID NOs:264, 265, and 266, respectively, andwherein the VL when paired with a VH comprising the amino acid sequenceset forth in SEQ ID NO:259 binds to TDP-43.
 10. The cDNA of claim 9,wherein the polypeptide comprises: an immunoglobulin heavy chain orfragment thereof comprising a VH with the amino acid sequence set forthin SEQ ID NO:10; an immunoglobulin heavy chain or fragment thereofcomprising a VH with the amino acid sequence set forth in SEQ ID NO:130;an immunoglobulin heavy chain or fragment thereof comprising a VH withthe amino acid sequence set forth in SEQ ID NO:259; an immunoglobulinlight chain or fragment thereof comprising a VL with the amino acidsequence set forth in SEQ ID NO:14; an immunoglobulin light chain orfragment thereof comprising a VL with the amino acid sequence set forthin SEQ ID NO:134; or an immunoglobulin light chain or fragment thereofcomprising a VL with the amino acid sequence set forth in SEQ ID NO:263.11. An expression vector comprising a heterologous promoter operablylinked to a polynucleotide encoding a polypeptide comprising: animmunoglobulin heavy chain or a fragment thereof comprising a heavychain variable region (VH) comprising complementarity determiningregions (CDRs) 1, 2, and 3 with the amino acid sequences set forth inSEQ ID NOs:11, 12, and 13, respectively, and wherein the VH when pairedwith a light chain variable region (VL) comprising the amino acidsequence set forth in SEQ ID NO:14 binds to TDP-43; or an immunoglobulinlight chain or a fragment thereof comprising a VL comprising CDRs 1, 2,and 3 with the amino acid sequences set forth in SEQ ID NOs:15, 16, and17, respectively, and wherein the VL when paired with a VH comprisingthe amino acid sequence set forth in SEQ ID NO:10 binds to TDP-43. 12.The expression vector of claim 11, wherein the polypeptide comprises: animmunoglobulin heavy chain or fragment thereof comprising a VH with theamino acid sequence set forth in SEQ ID NO:10; or an immunoglobulinlight chain or fragment thereof comprising a VL with the amino acidsequence set forth in SEQ ID NO:14.
 13. The expression vector of claim11, wherein the expression vector is a plasmid, phage, or virus.
 14. Theexpression vector of claim 13, wherein the expression vector is aretrovirus.
 15. The expression vector of claim 11, wherein theheterologous promoter is a cytomegalovirus, simian virus 40, orretroviral promoter.
 16. The expression vector of claim 11, wherein theheterologous promoter is the cytomegalovirus immediate early promoter.17. The expression vector of claim 11, wherein the polypeptide comprisesa signal peptide.
 18. The expression vector of claim 17, wherein thesignal peptide is a heterologous signal peptide.
 19. An expressionvector comprising: a first polynucleotide encoding a first polypeptidecomprising an immunoglobulin heavy chain or a fragment thereofcomprising a heavy chain variable region (VH) comprising complementaritydetermining regions (CDRs) 1, 2, and 3 with the amino acid sequences setforth in SEQ ID NOs:11, 12, and 13, respectively; and a secondpolynucleotide encoding a second polypeptide comprising animmunoglobulin light chain or a fragment thereof comprising a VLcomprising CDRs 1, 2, and 3 with the amino acid sequences set forth inSEQ ID NOs:15, 16, and 17, respectively, wherein the immunoglobulinheavy chain or fragment thereof when paired with the immunoglobulinlight chain or fragment thereof forms an anti-human TDP-43 antibody orTDP-43-binding fragment thereof, and wherein the expression vector is aplasmid, phage, or virus.
 20. The expression vector of claim 19, whereinthe expression vector is a retrovirus.
 21. The expression vector ofclaim 19, wherein: the VH consists of the amino acid sequence set forthin SEQ ID NO:10; and the VL consists of the amino acid sequence setforth in SEQ ID NO:14.
 22. The expression vector of claim 19, whereinthe first polypeptide comprises a human IgG1 heavy chain constantregion.
 23. The expression vector of claim 19, wherein the secondpolypeptide comprises a human kappa light chain constant region.
 24. Anisolated host cell comprising: a first expression vector comprising afirst heterologous promoter operably linked to a first polynucleotideencoding a first polypeptide comprising an immunoglobulin heavy chain ora fragment thereof comprising a heavy chain variable region (VH)comprising VH complementarity determining regions (CDRs) 1, 2, and 3with the amino acid sequences set forth in SEQ ID NOs:11, 12, and 13,respectively; and a second expression vector comprising a secondheterologous promoter operably linked to a second polynucleotideencoding a second polypeptide comprising an immunoglobulin light chainor a fragment thereof comprising a light chain variable region (VL)comprising VL CDRs 1, 2, and 3 with the amino acid sequences set forthin SEQ ID NOs:15, 16, and 17, respectively, wherein the immunoglobulinheavy chain or fragment thereof when paired with the immunoglobulinlight chain or fragment thereof forms an anti-TDP-43 antibody or humanTDP-43-binding fragment thereof.
 25. The isolated host cell of claim 24,wherein the first and second heterologous promoters are acytomegalovirus, simian virus 40, or retroviral promoter.
 26. Theisolated host cell of claim 24, wherein the first and secondheterologous promoters are the cytomegalovirus immediate early promoter.27. The isolated host cell of claim 24, wherein the first and secondexpression vectors are a plasmid, phage, or virus.
 28. The isolated hostcell of claim 24, wherein the first and second expression vectors are aretrovirus.
 29. The isolated host cell of claim 24, wherein: the VHconsists of the amino acid sequence set forth in SEQ ID NO:10; and theVL consists of the amino acid sequence set forth in SEQ ID NO:14. 30.The isolated host cell of claim 24, wherein the first polypeptidecomprises a human IgG1 heavy chain constant region.
 31. The isolatedhost cell of claim 24, wherein the second polypeptide comprises a humankappa light chain constant region.
 32. A method for preparing ananti-human TDP-43 antibody or human TDP-43-binding fragment thereof, themethod comprising: culturing the isolated host cell of claim 24 in acell culture under conditions that allow for expression of theanti-human TDP-43 antibody or human TDP-43-binding fragment thereof; andisolating the anti-human TDP-43 antibody or human TDP-43-bindingfragment thereof from the cell culture.
 33. The method of claim 32,further comprising formulating the anti-human TDP-43 antibody or humanTDP-43-binding fragment thereof into a sterile pharmaceuticalcomposition suitable for administration to a human subject.
 34. Themethod of claim 33, wherein the pharmaceutical composition is suitablefor intravenous or subcutaneous administration.
 35. The method of claim32, wherein: the VH consists of the amino acid sequence set forth in SEQID NO:10; and the VL consists of the amino acid sequence set forth inSEQ ID NO:14.
 36. The method of claim 35, wherein the first polypeptidecomprises a human IgG1 heavy chain constant region and the secondpolypeptide comprises a human kappa light chain constant region.
 37. Anexpression vector comprising a heterologous promoter operably linked toa polynucleotide encoding a polypeptide comprising: an immunoglobulinheavy chain or a fragment thereof comprising a heavy chain variableregion (VH) comprising complementarity determining regions (CDRs) 1, 2,and 3 with the amino acid sequences set forth in SEQ ID NOs:131, 132,and 133, respectively, and wherein the VH when paired with a light chainvariable region (VL) comprising the amino acid sequence set forth in SEQID NO:134 binds to TDP-43; or an immunoglobulin light chain or afragment thereof comprising a VL comprising CDRs 1, 2, and 3 with theamino acid sequences set forth in SEQ ID NOs:135, 136, and 137,respectively, and wherein the VL when paired with a VH comprising theamino acid sequence set forth in SEQ ID NO:130 binds to TDP-43.
 38. Theexpression vector of claim 37, wherein the polypeptide comprises: animmunoglobulin heavy chain or fragment thereof comprising a VH with theamino acid sequence set forth in SEQ ID NO:130; or an immunoglobulinlight chain or fragment thereof comprising a VL with the amino acidsequence set forth in SEQ ID NO:134.
 39. The expression vector of claim37, wherein the expression vector is a plasmid, phage, or virus.
 40. Theexpression vector of claim 39, wherein the expression vector is aretrovirus.
 41. The expression vector of claim 37, wherein theheterologous promoter is a cytomegalovirus, simian virus 40, orretroviral promoter.
 42. The expression vector of claim 37, wherein theheterologous promoter is the cytomegalovirus immediate early promoter.43. The expression vector of claim 37, wherein the polypeptide comprisesa signal peptide.
 44. The expression vector of claim 43, wherein thesignal peptide is a heterologous signal peptide.
 45. An expressionvector comprising: a first polynucleotide encoding a first polypeptidecomprising an immunoglobulin heavy chain or a fragment thereofcomprising a heavy chain variable region (VH) comprising complementaritydetermining regions (CDRs) 1, 2, and 3 with the amino acid sequences setforth in SEQ ID NOs:131, 132, and 133, respectively; and a secondpolynucleotide encoding a second polypeptide comprising animmunoglobulin light chain or a fragment thereof comprising a VLcomprising CDRs 1, 2, and 3 with the amino acid sequences set forth inSEQ ID NOs:135, 136, and 137, respectively, wherein the immunoglobulinheavy chain or fragment thereof when paired with the immunoglobulinlight chain or fragment thereof forms an anti-human TDP-43 antibody orTDP-43-binding fragment thereof, and wherein the expression vector is aplasmid, phage, or virus.
 46. The expression vector of claim 45, whereinthe expression vector is a retrovirus.
 47. The expression vector ofclaim 45, wherein: the VH consists of the amino acid sequence set forthin SEQ ID NO:130; and the VL consists of the amino acid sequence setforth in SEQ ID NO:134.
 48. The expression vector of claim 45, whereinthe first polypeptide comprises a human IgG1 heavy chain constantregion.
 49. The expression vector of claim 45, wherein the secondpolypeptide comprises a human kappa light chain constant region.
 50. Anisolated host cell comprising: a first expression vector comprising afirst heterologous promoter operably linked to a first polynucleotideencoding a first polypeptide comprising an immunoglobulin heavy chain ora fragment thereof comprising a heavy chain variable region (VH)comprising VH complementarity determining regions (CDRs) 1, 2, and 3with the amino acid sequences set forth in SEQ ID NOs:131, 132, and 133,respectively; and a second expression vector comprising a secondheterologous promoter operably linked to a second polynucleotideencoding a second polypeptide comprising an immunoglobulin light chainor a fragment thereof comprising a light chain variable region (VL)comprising VL CDRs 1, 2, and 3 with the amino acid sequences set forthin SEQ ID NOs:135, 136, and 137, respectively, wherein theimmunoglobulin heavy chain or fragment thereof when paired with theimmunoglobulin light chain or fragment thereof forms an anti-TDP-43antibody or human TDP-43-binding fragment thereof.
 51. The isolated hostcell of claim 50, wherein the first and second heterologous promotersare a cytomegalovirus, simian virus 40, or retroviral promoter.
 52. Theisolated host cell of claim 50, wherein the first and secondheterologous promoters are the cytomegalovirus immediate early promoter.53. The isolated host cell of claim 50, wherein the first and secondexpression vectors are a plasmid, phage, or virus.
 54. The isolated hostcell of claim 50, wherein the first and second expression vectors are aretrovirus.
 55. The isolated host cell of claim 50, wherein: the VHconsists of the amino acid sequence set forth in SEQ ID NO:130; and theVL consists of the amino acid sequence set forth in SEQ ID NO:134. 56.The isolated host cell of claim 50, wherein the first polypeptidecomprises a human IgG1 heavy chain constant region.
 57. The isolatedhost cell of claim 50, wherein the second polypeptide comprises a humankappa light chain constant region.
 58. A method for preparing ananti-human TDP-43 antibody or human TDP-43-binding fragment thereof, themethod comprising: culturing the isolated host cell of claim 50 in acell culture under conditions that allow for expression of theanti-human TDP-43 antibody or human TDP-43-binding fragment thereof; andisolating the anti-human TDP-43 antibody or human TDP-43-bindingfragment thereof from the cell culture.
 59. The method of claim 58,further comprising formulating the anti-human TDP-43 antibody or humanTDP-43-binding fragment thereof into a sterile pharmaceuticalcomposition suitable for administration to a human subject.
 60. Themethod of claim 59, wherein the pharmaceutical composition is suitablefor intravenous or subcutaneous administration.
 61. The method of claim58, wherein: the VH consists of the amino acid sequence set forth in SEQID NO:130; and the VL consists of the amino acid sequence set forth inSEQ ID NO:134.
 62. The method of claim 61, wherein the first polypeptidecomprises a human IgG1 heavy chain constant region and the secondpolypeptide comprises a human kappa light chain constant region.
 63. Anexpression vector comprising a heterologous promoter operably linked toa polynucleotide encoding a polypeptide comprising: an immunoglobulinheavy chain or a fragment thereof comprising a heavy chain variableregion (VH) comprising complementarity determining regions (CDRs) 1, 2,and 3 with the amino acid sequences set forth in SEQ ID NOs:260, 261,and 262, respectively, and wherein the VH when paired with a light chainvariable region (VL) comprising the amino acid sequence set forth in SEQID NO:263 binds to TDP-43; or an immunoglobulin light chain or afragment thereof comprising a VL comprising CDRs 1, 2, and 3 with theamino acid sequences set forth in SEQ ID NOs:264, 265, and 266,respectively, and wherein the VL when paired with a VH comprising theamino acid sequence set forth in SEQ ID NO:259 binds to TDP-43.
 64. Theexpression vector of claim 63, wherein the polypeptide comprises: animmunoglobulin heavy chain or fragment thereof comprising a VH with theamino acid sequence set forth in SEQ ID NO:259; or an immunoglobulinlight chain or fragment thereof comprising a VL with the amino acidsequence set forth in SEQ ID NO:263.
 65. The expression vector of claim63, wherein the expression vector is a plasmid, phage, or virus.
 66. Theexpression vector of claim 65, wherein the expression vector is aretrovirus.
 67. The expression vector of claim 63, wherein theheterologous promoter is a cytomegalovirus, simian virus 40, orretroviral promoter.
 68. The expression vector of claim 63, wherein theheterologous promoter is the cytomegalovirus immediate early promoter.69. The expression vector of claim 63, wherein the polypeptide comprisesa signal peptide.
 70. The expression vector of claim 69, wherein thesignal peptide is a heterologous signal peptide.
 71. An expressionvector comprising: a first polynucleotide encoding a first polypeptidecomprising an immunoglobulin heavy chain or a fragment thereofcomprising a heavy chain variable region (VH) comprising complementaritydetermining regions (CDRs) 1, 2, and 3 with the amino acid sequences setforth in SEQ ID NOs:260, 261, and 262, respectively; and a secondpolynucleotide encoding a second polypeptide comprising animmunoglobulin light chain or a fragment thereof comprising a VLcomprising CDRs 1, 2, and 3 with the amino acid sequences set forth inSEQ ID NOs:264, 265, and 266, respectively, wherein the immunoglobulinheavy chain or fragment thereof when paired with the immunoglobulinlight chain or fragment thereof forms an anti-human TDP-43 antibody orTDP-43-binding fragment thereof, and wherein the expression vector is aplasmid, phage, or virus.
 72. The expression vector of claim 71, whereinthe expression vector is a retrovirus.
 73. The expression vector ofclaim 71, wherein: the VH consists of the amino acid sequence set forthin SEQ ID NO:259; and the VL consists of the amino acid sequence setforth in SEQ ID NO:263.
 74. The expression vector of claim 71, whereinthe first polypeptide comprises a human IgG1 heavy chain constantregion.
 75. The expression vector of claim 71, wherein the secondpolypeptide comprises a human kappa light chain constant region.
 76. Anisolated host cell comprising: a first expression vector comprising afirst heterologous promoter operably linked to a first polynucleotideencoding a first polypeptide comprising an immunoglobulin heavy chain ora fragment thereof comprising a heavy chain variable region (VH)comprising VH complementarity determining regions (CDRs) 1, 2, and 3with the amino acid sequences set forth in SEQ ID NOs:260, 261, and 262,respectively; and a second expression vector comprising a secondheterologous promoter operably linked to a second polynucleotideencoding a second polypeptide comprising an immunoglobulin light chainor a fragment thereof comprising a light chain variable region (VL)comprising VL CDRs 1, 2, and 3 with the amino acid sequences set forthin SEQ ID NOs:264, 265, and 266, respectively, wherein theimmunoglobulin heavy chain or fragment thereof when paired with theimmunoglobulin light chain or fragment thereof forms an anti-TDP-43antibody or human TDP-43-binding fragment thereof.
 77. The isolated hostcell of claim 76, wherein the first and second heterologous promotersare a cytomegalovirus, simian virus 40, or retroviral promoter.
 78. Theisolated host cell of claim 76, wherein the first and secondheterologous promoters are the cytomegalovirus immediate early promoter.79. The isolated host cell of claim 76, wherein the first and secondexpression vectors are a plasmid, phage, or virus.
 80. The isolated hostcell of claim 76, wherein the first and second expression vectors are aretrovirus.
 81. The isolated host cell of claim 76, wherein: the VHconsists of the amino acid sequence set forth in SEQ ID NO:259; and theVL consists of the amino acid sequence set forth in SEQ ID NO:263. 82.The isolated host cell of claim 76, wherein the first polypeptidecomprises a human IgG1 heavy chain constant region.
 83. The isolatedhost cell of claim 76, wherein the second polypeptide comprises a humankappa light chain constant region.
 84. A method for preparing ananti-human TDP-43 antibody or human TDP-43-binding fragment thereof, themethod comprising: culturing the isolated host cell of claim 76 in acell culture under conditions that allow for expression of theanti-human TDP-43 antibody or human TDP-43-binding fragment thereof; andisolating the anti-human TDP-43 antibody or human TDP-43-bindingfragment thereof from the cell culture.
 85. The method of claim 84,further comprising formulating the anti-human TDP-43 antibody or humanTDP-43-binding fragment thereof into a sterile pharmaceuticalcomposition suitable for administration to a human subject.
 86. Themethod of claim 85, wherein the pharmaceutical composition is suitablefor intravenous or subcutaneous administration.
 87. The method of claim84, wherein: the VH consists of the amino acid sequence set forth in SEQID NO:259; and the VL consists of the amino acid sequence set forth inSEQ ID NO:263.
 88. The method of claim 87, wherein the first polypeptidecomprises a human IgG1 heavy chain constant region and the secondpolypeptide comprises a human kappa light chain constant region.